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 Transformation
of
Human Epithelial Cells:

Molecular and Oncogenetic

Mechanisms

Editors

George E. Milo, M.S., Ph.D.

Professor, Department of Medical Biochemistry
Director of Carcinogenesis and Molecular Toxicology
Comprehensive Cancer Center
The Ohio State University
Columbus, Ohio

Bruce C. Casto, M.S., Sc.D.

Director of Research
Environmental Health and Research Testing, Inc.
Research Triangle Park, North Carolina

Charles F. Shuler, D.M.D., Ph.D.

Assistant Professor, Center for Craniofacial Molecular Biology
University of Southern California
Los Angeles, California

CRC
\CP* J Taylor & Francis Group
^***^/ Boca Raton London New York

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First published 1992 by CRC Press
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 PREFACE

Several years ago, in the 1960s and 1970s, there were but a few human
cell lines available to study human cell carcinogenesis. At that time the
"Hayflick" hypothesis suggested that human cells cultured in vitro have a
finite life-span of approximately 60 PDs; little was known of the "real"
relationship between the limited life-span of the human fibroblast in vitro and
aging in vivo. This area of research piqued the interest of the scientific
community because the expression of cancer was thought to be an escape of
the neoplastic phenotype from the limited proliferative potential, i.e., the
finite life-span. However, over the years many human cancers were deter-
mined to be of an epithelial origin, and it was not possible to isolate, except
on a rare occurrence, stable tumor phenotypes in vitro that exhibited infinite
life-spans. Consistently heterogenous human tumor phenotypes either ceased
to proliferate or terminally differentiated in vitro. Rarely did we observe in
a routine fashion an escape from a limited life-span to a phenotype with an
unrestricted proliferative potential. With the advent of collagen-coated plastic
substratum, feeder layers, "quasi"-chemically defined growth media con-
taining the likes of pituitary extracts, and growth factors, epithelial cells from
different human tissues could be isolated and cultured for limited defined
periods in vitro. Still the concept of the limited life-span of normal cells in
vitro persisted and the cancer cell was thought to be an escape from a limited
life-span. This loss of growth control and extension of life-span are discussed
in Chapter 5 by Johng S. Rhim, one of the pioneers in the field of SV-40T-
induced immortalization of human cells. We have included also a chapter on
another developing field that will dramatically impact the field of growth
control and life-span. Chapter 2 examines how autocrine and paracrine factors
elicit proliferative growth responses in normal and transformed cells. In the
1970s, Heppner recognized along with others that epithelial tumors were
heterogenous in cellular composition. Since that time we have learned how
to identify different phenotypes from the epithelial tumors. Because of the
recent achievements by Bert Vogelstein in the late 1980s of identifying, in
colon carcinoma tumors, cells that occupy different stages of progression and
the discovery by Patricia Steggs and Lance Liotta in the late 1980s of the
molecular events that were associated with expression of the metastatic stage,
we can now follow the events of metabolism of putative human carcinogens,
DNA-adduct binding, early, middle and late stages of progression of initiated
cells, anchorage-independent growth, identification of expression of altered
phenotypes, and identification of expression of tumorigenic and metastatic
phenotypes. If we agree that tumors are clonal in origin, then we need to
understand that tumor heterogeneity may be a function of the composition of
mixed phenotypes. Some of these questions comprise the reasons this book
and the contributors were assembled to address the stages from metabolism
to metastasis of epithelial cells associated with epithelial tumors.
 In Chapter 1, James R. Smith addresses the role immortalization plays
in cancer and how resistant human cells are to spontaneous immortalization.
There has been an attempt to link the loss of expression of a finite life-span
to the change in genetic program of either the 1st or the 4th chromosome. A
defect in these chromosomes putatively can give rise to the expression of a
cellular phenotype that exhibits immortalization. However, at present (in these
authors' opinion) the question of quiescence vs. senescence in human cells
has not been answered by these experiments. The loss of senescent control
can lead to immortalization; the loss of control of expression of quiescence
cannot. However, it is recognized that the transient down-regulation of the
cyclin gene or the permanent interruption of this gene function may play a
pivotal role in the two processes. The next several chapters discuss the stages
of multi-stage carcinogenesis in different epithelial cell systems. Moreover,
correlative stages of progression in different carcinogen-transformed epithelial
cell systems are compared and contrasted. The last few chapters — 10, 11,
and 12 — discuss molecular controlling mechanisms that are involved in the
control of expression of stages of progression, e.g., the role of oncogenes
and their interactiveness with the suppressor genes. In particular, the role of
ras gene mutations and suppressor gene interaction with these activated on-
cogenes in tumorigenic cells in controlling the progression of initiated cells
into tumor cells is presented. The promotion stage in human cell carcino-
genesis is a silent stage and has not been observed experimentally, and many
attempts to discover this stage have led to failure.
In Chapter 13 we compare and contrast the stages from metabolism, DNA-
adduct formation, anchorage-independent tumor growth, and regression be-
tween epithelial cells and fibroblast (see also Transformation of Human Dip-
loid Fibroblast: Molecular and Genetic Mechanisms, Milo, G. E. and Casto,
B . C . , Eds., CRC Press, 1990). Lastly, an invitation was extended to Juergen
R. Vielkind to contribute a guest chapter discussion on the nature of growth
control in osteichthyes. Our reasons for including this chapter are to develop
and understand the biological significances of the conservation of suppressor
gene function phylogenetically from lower animals up to humans and to
understand the role of some of these genes in more primitive animal systems.
It was our intention to present to the scientific community a forum for
focusing on the utility of how human epithelial cells as systems can be used
to examine how environmental xenobiotics can be metabolized to reactive
metabolites that can react with critical sites on the genomic DNA that lead
to the expression of an early stage of a transformed phenotype. Later in the
book we have focused on how current dogma in suppressor gene-oncogene
interaction is insufficient to totally explain the existence of many diverse
phenotypes in a heterogenous spontaneous epithelial cell tumor. Moreover,
the concept of plasticity — the transient expression of a tumorigenic phen-
otype — is not explained only by the presence of mutations in critical sites
of activated oncogenes or mutations in suppressor genes. This treatise is an
attempt to collate many of the scientific results, significant scientific concepts,
and laboratory efforts from active investigators in this field of environmentally
induced human epithelial cell cancer.

George E. Milo
Bruce C. Casto
Charles F. Shuler
 THE EDITORS

George E. Milo, B.A., M.S., Ph.D., is Professor of Medical Biochem-

istry in the College of Medicine and Program Director of Molecular Envi-
ronmental Health in the Comprehensive Cancer Center at The Ohio State
University in Columbus.
Dr. Milo has actively pursued research in the discipline of human cell
carcinogenesis since 1970. He published the first report on the transformation
of human fibroblast cells in 1978 in Nature and the first report on the trans-
formation of human epithelial cells in Cancer Research in 1981. Again in
1990, along with Dr. Charles Shuler, he published a new concept on the
isolation and identification of a plastic anchorage-independent growth of a
nontumorigenic phenotype that can be transiently converted to a tumorigenic
and metastatic phenotype. This article was published in the Proceedings of
the National Academy of Science (U.S.A.). His National Institutes of Health
(NIH)-supported postdoctoral training at the Roswell Park Memorial Institute
in Cancer Research (Buffalo, NY) served him well as a stepping stone to
begin his career in human cell carcinogenesis.
Dr. Milo is a member of the Society for Toxicology — Molecular Tox-
icology Division, the American Association for Cancer Research, the Amer-
ican Society for Biochemistry and Molecular Biology, the American Society
for Cell Biology, and the International Society for the Study of Xenobiotics.
He has published in excess of 130 publications in the discipline of human
cell carcinogenesis. He has written several chapters for different books on
the subject and has served as an ad hoc reviewer for the National Institutes
of Health — National Cancer Institute (NIH-NCI) in the discipline, as a
reviewer and Chairperson for the U.S. Environmental Protection Agency
Extramural Health Effects Research Review Panel, as a Chairperson for the
U.S. Environmental Protection Agency ''Health" Research Centers program,
and as a reviewer on the NIH-NCI Parent Preclinical Pharmacology Program
Panel. He has received many grants from the National Institutes of Health
— National Cancer Institute and National Institute of Environmental Health
Science — and from the U.S. Environmental Protection Agency — Health
Effects Research. He also edited, along with Dr. Bruce C. Casto, Transfor-
mation of Human Fibroblasts: Molecular and Genetic Mechanisms, published
by CRC Press in 1990.
His present area of interest is to investigate how exposure to environmental
xenobiotics alters human gene function.

Bruce C. Casto, M.S., Sc.D., is Director of Research for Environmental

Health and Research Testing, Inc. in Research Triangle Park, North Carolina.
Dr. Casto received training in microbiology and virology at The Ohio State
University and the University of Pittsburgh. During this time, research was
conducted in the areas of viral receptors, oncolytic viruses, and adeno-as-
sociated viruses. While at the University of Pittsburgh, Dr. Casto discovered
the defective nature of AAV-1 and its dependence on adenovirus for repli-
cation. He was an assistant member at the Institute for Biomedical Research
— American Medical Association, professor of microbiology at Rush Medical
School, senior scientist at BioLabs, Inc., and research director for health
effects at Northrop Environmental Sciences. Dr. Casto's major area of re-
search is chemical carcinogenesis, especially the enhancement of viral trans-
formation by chemical carcinogens and the chemical transformation of mam-
malian cells in vitro.

Charles F. Shuler, D.M.D., Ph.D., is Assistant Professor in the Center

for Craniofacial Molecular Biology at the University of Southern California
School of Dentistry.
Dr. Shuler received his dental degree from the Harvard University School
of Dental Medicine, his Oral Pathology training at the University of Min-
nesota, and his Ph.D. in Experimental Pathology from the University of
Chicago.
Dr. Shuler is a member of the American Society for Cell Biology, the
American Association for Dental Research, the American Association for
Advancement of Science, and the American Academy of Oral Pathology. He
has served as an Associate Editor of the Journal of Oral Pathology.
His current areas of research interest include mechanisms of epithelial
differentiation, especially during the development of the secondary palate in
utero, and human cell transformation and tumorigenesis.
 CONTRIBUTORS

William M. Baird, Ph.D. Frank C. Cuttitta, Ph.D.

Glenn L. Jenkins Professor of Biomarker and Prevention
Medicinal Chemistry Research Branch
and Purdue Cancer Center NCI-Navy Medical Oncology
Purdue University Branch
West Lafayette, Indiana Biotherapy Section
National Naval Medical Center
Linda L. Barrett, M.S. National Cancer Institute
School of Medicine and Department of Medicine
East Carolina University Uniformed Services University of
Greenville, North Carolina the Health Sciences
Bethesda, Maryland
Michael J. Birrer, M.D., Ph.D.
NCI-Navy Medical Oncology
Curtis C. Harris, M.D.
Branch
Laboratory of Human
Clinical Oncology Program
Carcinogenesis
Division of Cancer Treatment
National Cancer Institute
National Cancer Institute
Bethesda, Maryland
Bethesda, Maryland
David G. Kaufman, M.D.,
Tammela Butler, B.S.
Ph.D.
Department of Toxicology
Department of Pathology
University of North Carolina
University of North Carolina
Chapel Hill, North Carolina
School of Medicine
Chapel Hill, North Carolina
Charleata A. Carter, Ph.D.
Experimental Carcinogenesis and
Mutagenesis Branch Hudson H. S. Lau, Ph.D.
National Institutes of Department of Medicinal
Environmental Health Services Chemistry Pharmacognosy
Research Triangle Park, North Purdue University
Carolina West Lafayette, Indiana

Bruce C. Casto, M.S., Sc.D.

Caroline H. Laundon, Ph.D.
Environmental Health & Research
GeneCare
Testing, Inc.
Chapel Hill, North Carolina
Research Triangle Park, North
Carolina
Teresa A. Lehman, Ph.D.
Dharam P. Chopra, Ph.D. Laboratory of Human
Institute of Chemical Toxicology Carcinogenesis
Wayne State University National Cancer Institute
Detroit, Michigan Bethesda, Maryland
George E. Milo, Ph.D. James R. Smith, Ph.D.
Department of Medical Roy M. & Phyllis Gough
Biochemistry Huffington Center on Aging
and Department of Molecular and Division of Molecular
Environmental Health of the Virology
Comprehensive Cancer Center Baylor College of Medicine
The Ohio State University Houston, Texas
Columbus, Ohio
Martha R. Stampfer, Ph.D.
Zenya Naito, M.D., Ph.D. Department of Cell and Molecular
Medical Technology Biology
Yokosuka National Hospital Lawrence Berkeley Laboratory
Yokosuka, Kanagawa, Japan University of California
Berkeley, California
Johng Sik Rhim, M.D.
Department of Radiation Medicine Gary M. Stoner, Ph.D.
Georgetown University School of Experimental Pathology
Medicine Department of Pathology
Washington, D.C. Medical College of Ohio
and Laboratory of Cellular and Toledo, Ohio
Molecular Biology
National Cancer Institute Juergen R. Vielkind, Ph.D.
Bethesda, Maryland Department of Cancer
Endocrinology
Clifford A. Rinehart, Ph.D. British Columbia Cancer Agency
Department of Pathology and Department of Pathology
University of North Carolina University of British Columbia
Chapel Hill, North Carolina Vancouver, British Columbia,
Canada
Charles F. Shuler, D.M.D.,
Ph.D. Li Hui Xu, M.D.
Center for Craniofacial Molecular Department of Pathology
Biology University of North Carolina
School of Dentistry Chapel Hill, North Carolina
University of Southern California
Los Angeles, California Paul Yaswen
Department of Cell and Molecular
Jill Siegfried, Ph.D. Biology
Department of Pharmacology Lawrence Berkeley Laboratory
University of Pittsburgh University of California
Pittsburgh, Pennsylvania Berkeley, California
 TABLE OF CONTENTS

Chapter 1
In Vitro Cellular Aging and Immortalization 1
James R. Smith

Chapter 2
Detection of Growth Factor Effects and Expression in Normal and
Neoplastic Human Bronchial Epithelial Cells 13
Jill M. Siegfried, Michael J. Birrer, and Frank C. Cuttitta

Chapter 3
Human Cell Metabolism and DNA Adduction of Polycyclic Aromatic
Hydrocarbons 31
Hudson H. S. Lau and William M. Baird

Chapter 4
Human Esophageal Epithelial Cells: Immortalization and In Vitro
Transformation 67
Gary D. Stoner, Zenya Naito, and George E. Milo

Chapter 5
Transformation of Human Endometrial Stromal Cells In Vitro 85
Clifford A. Rinehart, Charleata A. Carter, Li Hui Xu, Linda L. Barrett,
Tammela Butler, Caroline H. Laundon, and David G. Kaufman

Chapter 6
Factors Influencing Growth and Differentiation of Normal and
Transformed Human Mammary Epithelial Cells in Culture 117
Martha R. Stampfer and Paul Yaswen

Chapter 7
Transformation of Colon Epithelial Cells 141
Dharam P. Chopra

Chapter 8
Multistep Carcinogenesis and Human Epithelial Cells 169
Johng S. Rhim

Chapter 9
Morphologic and Molecular Characterizations of Plastic Tumor Cell
Phenotypes 211
Charles F. Shuler and George E. Milo
Chapter 10
Oncogene and Tumor Suppressor Gene Involvement in Human Lung
Carcinogenesis 235
Teresa S. Lehman and Curtis C. Harris

Chapter 11
Events of Tumor Progression Associated with Carcinogen Treatment
of Epithelial and Fibroblast Compared with Mutagenic Events 261
George E. Milo and Bruce C. Casto

Chapter 12
Progression from Pigment Cell Patterns to Melanomas in Platyfish-
Swordtail Hybrids — Multiple Genetic Changes and a Theme for
Tumorigenesis 285
Juergen R. Vielkind

Index 303
Smith 1

Chapter 1

IN VITRO CELLULAR AGING AND IMMORTALIZATION

James R. Smith

TABLE OF CONTENTS

I. Introduction 2

II. In Vitro Cellular Aging is Dominant in Somatic Cell Hybrids 3

A. Limited In Vitro Life-Span of Normal Cells 3
B. Hybrids between Normal and Immortal Cells 3
C. Fusion of Immortal Cells with Immortal Cell Lines 4
D. Microcell Hybrid Experiments 6

III. Cellular Aging Is an Active Process 6

A. Heterokaryon Experiments 6
B. Membrane-Associated DNA Synthesis Inhibitors of
Senescent Cells 7
C. Inhibition of DNA Synthesis by Poly (A) + RNA 8

IV. Discussion 9

References 10
2 Transformation of Human Epithelial Cells

I. INTRODUCTION

Carcinogenesis has become widely accepted as a multistep process2 (see

References 1 and 2 for recent reviews). A number of events have to occur
in order for cells to become cancerous. In many cases, if not all, cellular
immortalization is one of these steps and is an obligatory process.3 Normal
human diploid cells go through various numbers of population doublings
(PDs), depending on the age of the donor and the origin of the tissue from
which the cells are derived.4"6 In most cases, cells from adult donors go
through fewer PDs than cells from young or embryonic donors.6 The number
of PDs that a culture can go through when derived from adult tissue is typically
20 to 30.5 Cancers generally are of clonal origin, and for a single cell to
produce a tumor 1 g in size requires approximately 30 cell divisions. Primary
tumors are not the major cause of problems in carcinogenesis because of the
possibility of surgical removal of the primary tumor and, hence, the threat
to the individual by that tumor. Indeed, metastasis is the crucial step that
causes carcinogenesis to be a life-threatening phenomenon. Metastases are
also generally of clonal origin and require a cell that has already gone through
a number of doublings in the primary tumor to undergo further doublings as
a metastatic growth in order to be significant. Cell growth, tumor regression,
and cell death are all normal parts of the processes of carcinogenesis. There-
fore, the number of PDs that cells have to go through in order to become
life-threatening may be more than 100 to 200. This range is clearly greater
than normal cells are able to go through, as evidenced by experiences with
human fibroblasts in tissue culture. 7 Other cells in the body may normally be
able to go through more doublings in vivo. However, at this time, the doubling
limit for most epithelial cells in situ is unknown. Therefore, it is reasonable
to assume that as part of the multistep process of carcinogenesis, cellular
immortalization is required for tumor progression and metastasis. The spon-
taneous immortalization of human cells in culture has never been observed.
However, this can be contrasted with the situation that we see in rodent cells,
particularly mouse and rat cells, in which spontaneous immortalization is the
rule rather than the exception. 810 When considering whether immortalization
may be necessary for tumor formation and metastasis, it is interesting to
compare the rates of tumor formation in rodents with those in humans. A
mouse weighs on the order of 10 g while humans weigh on the order of 100 kg,
and the mouse's life-span is approximately 1/30 that of a human, yet mice
very often have tumors during their 2V2- to 3-year life-span. Therefore, on
a per cell unit time basis, the rate of tumor formation in mice is 105 to 106
times the rate of tumor formation in humans. It seems likely that this incredibly
higher rate of tumor formation seen in mice compared to humans is due to
the much higher incidence of spontaneous immortalization of mouse cells
compared with human cells. Therefore, the study of cellular immortalization
and of mechanisms that limit the proliferative potential of normal human cells
in culture is of paramount importance in understanding the mechanisms of
carcinogenesis in humans.
Smith

II. IN VITRO CELLULAR AGING IS DOMINANT IN

SOMATIC CELL HYBRIDS

A. LIMITED IN VITRO LIFE-SPAN OF NORMAL CELLS

Swim and Parker were the first to show that human fibroblasts derived
from biopsies had a limited proliferative potential in culture. 11 Hay flick and
Moorehead in 1961 showed that these cells were karyotypically normal and
that normal cells derived from a large number of different individuals all had
a finite proliferative potential.4 They also showed that a major characteristic
of cells that were able to divide indefinitely, i.e., transformed immortal cells,
was an abnormal karyotype. In 1965, Hayflick proposed that the limited in
vitro proliferative potential of normal human fibroblasts in culture was a
manifestation of aging at the cellular level.5 More recently, it has been pro-
posed by O'Brien et al.3 that limited proliferative potential of normal cells
in vitro and also in vivo is a powerful tumor suppressor mechanism. The
observation of limited proliferative potential of normal cells in culture has
been repeated in hundreds of labs and thousands of cultures over the last 30
years.7 The proliferative potential of the cells depends on the age of the
donor,6 species of the donor,12 and the site of biopsy.6 Typically, human
embryonic cells will undergo 50 to 80 PDs before growth cessation, although
it has been reported that some cells are capable of going through approximately
100 PDs before proliferation stops.13 Cells from other species go through
fewer PDs than those from humans, the exception being the Galapagos turtle.14
The number of PDs that the cells are able to undergo is correlated with the
maximum life-span of the species.
Cells spontaneously immortalize at various rates, depending on the species
of origin of the cells. Human cells and chick cells have never been observed
to immortalize spontaneously, while rodent cells routinely immortalize in
culture and species such as bovine immortalize spontaneously only rarely.15
The mechanisms that lead to limited in vitro proliferative potential of normal
cells in culture is not understood. A number of investigations have been carried
out over the past 30 years to measure various biochemical, metabolic, and
structural parameters of these cells as they age in culture, and with very few
exceptions, which will be discussed later, no changes have been observed
that could account for the irreversible division cessation. Likewise, the process
by which cells escape the finite proliferative potential and become able to
divide without limit (immortalization) is not understood. In order to try to
understand the mechanisms operating in these processes, we and others have
undertaken a series of experiments discussed below.

B. HYBRIDS BETWEEN NORMAL AND IMMORTAL CELLS

The early work of Littlefield suggested that the limited proliferative po-
tential (the senescence phenotype) might be dominant in somatic cell hybrids
between senescent cells and young proliferating cells.16 However, the evi-
dence was not conclusive and the prevailing belief at that time was that cellular
4 Transformation of Human Epithelial Cells

immortalization was due to dominant changes in the cellular genome. Many

different ideas have been presented to try to explain the limited proliferation
of normal cells in culture. These can be broken into two main categories.
One category proposes that cells stop dividing because they accumulate dam-
age of various sorts, e.g., somatic cell mutations or errors in protein synthesis,
so that the error burden becomes so large that the cells are no longer able to
divide. The other category proposes some sort of genetic program that limits
the in vitro life-span of cells in culture. We thought that we might be able
to differentiate between these two broad categories of hypothesis by fusing
normal cells with immortal cells and determining whether the hybrids resulting
from that fusion had a limited in vitro life-span or were immortal. If normal
cells stopped dividing because they had accumulated a large amount of dam-
age, then one could argue that cells that are immortal have escaped from
limited proliferative potential because either they don't accumulate damage
at the same rate or they have evolved a mechanism to better cope with the
damage. Therefore, one might expect in hybrids that the phenotype of cellular
immortality would be dominant.
In the first set of fusions, we fused an immortal SV40-transformed cell
line with a normal cell line that was at the end of its in vitro life-span.17 We
observed that the hybrid colonies proliferated for various numbers of PDs and
then stopped dividing. About 70% of the colonies were able to go through
fewer than 8 PDs, while the other 30% were able to go through a range of
PDs varying from 30 to 60, but they all stopped dividing. We also showed
that all of the clones expressed the S V40 large T-antigen which is thought to
be the immortalizing agent for normal human diploid fibroblasts infected with
SV40 virus. In order to investigate the generality of this phenomenon, we
fused a number of different cell lines with normal human diploid fibroblasts
and observed the same results in all cases.18 The hybrids had finite proliferative
potential. In all the fusion experiments, we found that immortal variants arose
in the culture at a frequency of approximately 1 per 105 to 106 cells. This is
a much greater frequency of immortalization than that observed in normal
diploid fibroblasts. The tentative explanation for this is that in hybrids, chro-
mosomal segregation takes place and the hybrids lose a chromosome which
encodes a gene that causes the finite proliferative potential. Conclusions from
these experiments are that the limited life-span of normal cells in culture is
dominant over the phenotype of cellular immortality and that cells become
immortal because they lose some of the program that is necessary to impose
a limited proliferative potential on normal cells in culture.

C. FUSION OF IMMORTAL CELLS WITH OTHER IMMORTAL

CELL LINES
If cellular immortality results from recessive changes in the cellular ge-
nome, then we might expect that different defects could occur to render a
cell immortal. If that is the case, then fusion of cell lines having one defect
with cell lines having another defect could result in complementation, giving
Smith

a hybrid that has finite proliferative potential. On the other hand, fusion of
cell lines having the same defect would not result in complementation and
would give rise to hybrids that could divide indefinitely. Therefore, we would
predict that hybrids resulting from fusions among different immortal cell lines
would give two different kinds of results. In one case, some hybrids would
have a finite life-span and the other hybrids would have an indefinite life-
span. In a series of experiments, Pereira-Smith and Smith fused different cell
lines with each other and observed the proliferative phenotype (either finite
or indefinite),19 and assigned more than 30 different cell lines to four different
complementation groups. In order to begin the process of complementation
group assignment, one SV40-transformed cell line was chosen at random to
be representative of complementation group A. Other cell lines were fused
with it. Those that had an indefinite proliferative potential also assigned to
complementation group A; those that had a finite proliferative potential as-
signed to a different complementation group. Using HeLa as a prototypic cell
line for complementation group B, we repeated the process and assigned cell
lines to complementation group B. Cell lines were assigned to other com-
plementation groups in a similar fashion. This process involved numerous
cell fusions, and in no case did we find a cell line that assigned to more than
one complementation group. This indicated that the processes resulting in
cellular immortality were very rare, with no cell lines carrying two different
defects. We looked at a large number of different cell lines resulting from
different kinds of tumors, different cell types of origin, cell lines derived
from different embryonic layers, and cell lines that contained activated on-
cogenes, and in no cases did these parameters affect complementation group
assignment. The only parameter that did affect assignment was immortali-
zation by the SV40 large T-antigen. Seven out of eight of the SV40-immor-
talized cell lines assigned to complementation group A. One of the SV40 cell
lines failed to assign to complementation group A. The reason for this is not
known. We can speculate that the SV40 T-antigen was not the actual im-
mortalizing agent in this case, but was only coincidental in the transformation
process. The assignment of cell lines to different complementation groups
allows us to take a systematic approach to trying to understand what processes
might have occurred to result in cellular immortalization. We speculate at the
present time that those cell lines which assign to the same complementation
group have become immortalized by the same genetic defect. The case for
this interpretation is strengthened by the fact that almost all of the SV40-
transformed immortalized cell lines fall into the same complementation group.
However, it appears that not all DNA tumor viruses immortalize cells by the
same mechanism, because we found that cell lines immortalized by adeno,
papilloma, and herpes virus fell into different complementation groups. Efforts
are currently underway to find the genetic defect that leads to immortalization
in the case of SV40 T-antigen.
6 Transformation of Human Epithelial Cells

D. MICROCELL HYBRID EXPERIMENTS

The introduction of single normal human chromosomes into immortal
cell lines represents a considerable refinement over the techniques of somatic
cell hybridization involving whole cells discussed above. The use of microcell
hybrid techniques has allowed us to assign genes coding for normal cellular
aging processes to one particular human chromosome.
Ning et al.20'21 introduced chromosome 11 from a normal human cell line
into immortal cell lines representative of all four complementation groups.
They observed no effect on growth rate or the immortal phenotype of these
cells. There was some minor and variable effect on tumorigenicity when cells
carrying the intact human chromosome 11 were injected into nude mice.
There was, in some cases, suppression of tumorigenicity and, in other cases,
a delay in the formation of tumors.
Ning et al.22 further showed that introduction of a normal human chro-
mosome 4 into cell lines assigned to complementation group B restored the
phenotype of limited proliferative potential. However, when the human chro-
mosome 4 was introduced into cell lines assigning to the other complemen-
tation groups (A, C, and D), there was no decrease in proliferation potential
and the phenotype of immortality was retained. Thus, it seems clear that genes
on chromosome 4 code for some part of the genetic program that limits the
division potential of normal cells in culture. Disruption of these genes leads
to cells with an immortal phenotype. Sugawara et al.23 found a similar result
in studies in which they introduced the normal human chromosome 1 into
Chinese hamster cells. Human chromosome 1 was able to restore the cellular
aging phenotype in these immortal hamster cells. It remains to be seen whether
chromosome 1 plays a role in the immortalization of human cells.

III. CELLULAR AGING IS AN ACTIVE PROCESS

A. HETEROKARYON EXPERIMENTS
One of the first experiments that gave us an idea of the kinds of processes
that might be responsible for cellular senescence was performed by Norwood
et al.24 and independently by Stein and Yanishevsky. 25 They fused senescent
cells that had reached the end of their in vitro life-span with normal cells that
were still able to proliferate and asked whether the nuclei contained in the
heterokaryon were able to synthesize DNA. When senescent cells were fused
with young cells, it appeared that the senescent cell was able to suppress the
initiation of DNA synthesis in the young cell nucleus, i.e., neither the young
cell nucleus nor the senescent cell nucleus synthesized DNA in the hetero-
karyons up to 72 h after fusion. However, if young cells were fused with
each other, there was no decrease in the ability of the young cell nuclei to
synthesize DNA in the homodikaryon. From these results, it was concluded
that senescent cells produce an inhibitor of DNA synthesis which is able to
act in trans to inhibit the initiation of DNA synthesis in the young nucleus.
Furthermore, it has been shown that senescent cells, when fused with various
Smith

immortal cell lines,25 suppress DNA synthesis in the nucleus of the immortal
cell. This indicates that the inhibitor produced by senescent cells is able to
also inhibit the initiation of DNA synthesis in certain immortal cell lines.
However, other immortal cell lines,26 in particular those that have been im-
mortalized by DNA tumor viruses, e.g., SV40-transformed cells of HeLa
cells (HeLa is known now to have part of the herpes virus DNA integrated
into its genome), are able to induce DNA synthesis, in the short term, in the
senescent cell nucleus. This indicates that although senescent cells produce
an inhibitor of DNA synthesis, it is possible, through the intervention of DNA
tumor viruses, to temporarily override this inhibitor.27
Yanishevsky and Stein showed that the inhibitor of DNA synthesis present
in senescent cells could not interrupt ongoing DNA synthesis, but acted to
block the initiation of DNA synthesis. They found that if senescent cells were
fused to young cells that were more than 3 or 4 h from the S-phase, then
initiation of DNA synthesis was blocked. If they were closer to the S-phase,
then initiation of DNA synthesis was not blocked.28

B. MEMBRANE-ASSOCIATED DNA SYNTHESIS INHIBITORS OF

SENESCENT CELLS
Although the production of an inhibitor by senescent cells is a simple
and attractive explanation for the above results, there may be other expla-
nations. For example, if senescent nuclei were depleted of some factors that
were needed for induction of DNA synthesis and competed with the young
cell nucleus for those factors, then the concentration of positive regulatory
factors could fall below a critical threshold in the heterokaryons. In order
to rule out that possibility, we initiated experiments in which we prepared
enucleated cytoplasms from senescent cells and fused them to whole young
cells, and then asked whether the senescent cytoplasts could cause inhibition
of the initiation of DNA synthesis in the resulting cybrids. We found that
senescent cytoplasts were indeed capable of inhibiting the initiation of DNA
synthesis in young-cell cybrids.29"31 There was an approximately 50% decrease
in the number of young-cell nuclei synthesizing DNA in the senescent-young
cell cybrids compared to cybrids from young-cell cytoplasts fused with young
whole cells. We next asked the location of the inhibitor of DNA synthesis in
senescent cytoplasts. By treating the senescent cytoplasts with trypsin under
conditions that would limit the penetration of the trypsin into the cell and
limit intracellular damage by trypsin (4°C for 1 min), we were able to show
that the inhibitory activity resided on the outside surface of the membrane.
Further evidence for this conclusion was obtained by preparing membrane-
enriched fractions from senescent cells and adding them to young-cell cultures.
These membrane-enriched fractions were very effective in inhibiting the ini-
tiation of DNA synthesis in young-cell cultures.31-32 Furthermore, proteins
extracted from the membrane preparations and added directly to cultures of
young cells were also effective in inhibiting the initiation of DNA synthesis.31
We next examined the role of protein synthesis in the production of this
8 Transformation of Human Epithelial Cells

inhibitor by treating the cells with cyclohexamine or puromycin at concen-

trations which would inhibit protein synthesis by at least 90% . We found that
a relatively short treatment, approximately 2 h, with cyclohexamine or pu-
romycin was sufficient to eliminate the inhibitory activity from senescent
cells. Upon removal of cyclohexamine from the culture and incubation of the
cytoplasts in the absence of cyclohexamine, inhibitory activity was regained
in about 4 h,31 indicating that cytoplasts were still active and able to synthesize
DNA. Further, this indicated that the messenger RNA coding for the inhibitor
was relatively long-lived.

C. INHIBITION OF DNA SYNTHESIS BY POLY(A)+

In order to explore the feasibility of searching for cDNA clones coding
for the inhibitor, we microinjected poly(A)^ RNA from senescent cells into
young proliferation-competent cells to determine whether inhibitory activity
could be conferred by the messenger RNA. We found that it was strongly
inhibitory.33 By microinjecting different amounts of RNA into the cells, we
were able to calculate that the inhibitor RNA was present in relatively large
abundance (0. 1 to 1 % of the total messenger RNA consisted of inhibitor RNA
according to our calculations).
We also studied the possibility that nongrowing tissues, e.g., rat liver,
might produce an inhibitor. Rat liver was chosen because it can exist either
in a state of nonproliferation or in a state of proliferation. Lumpkin et al.34
found that nonregenerating liver RNA was able to block the initiation of DNA
synthesis when microinjected into young human fibroblasts, whereas the RNA
isolated from regenerating rat liver had no inhibitory activity; indeed, it had
a stimulatory activity. This raised the possibility of using rat liver as a source
of RNA to carry out syntheses and screening of a cDNA library to isolate
genes that were expressed in nonregenerating liver but not expressed in re-
generating liver. Inhibitory RNA has been hybrid selected by cDNA clones
isolated by differential screening of a cDNA library made from nonregener-
ating rat liver poly(A) + RNA. 35 However, these clones do not code for a
messenger RNA that is upregulated in senescent cells (unpublished data).
Other investigators have confirmed these results using RNA from human
liver36 and have extended them to show that RNA isolated from resting human
T-lymphocytes also will inhibit initiation of DNA synthesis when injected
into proliferation-competent human fibroblast cells.37 The results from these
microinjection experiments indicate that it would be feasible to search for
cDNAs coding for the inhibitory messenger RNA and the inhibitory protein.
Construction of cDNA libraries and screening by differential screening using
probes made from young cells and senescent cells is now underway in several
laboratories. Using this approach, various cDNA clones have been isolated;
however, as of yet, no clones have been isolated that have been shown to
code for the inhibitory activity expressed in senescent cells. Although the
cDNA cloning of inhibitor genes has not been fruitful to date, investigators
are still optimistic that straightforward ( + )/( — ) screening of cDNA libraries
Smith

produced from senescent cells or cells from premature aging syndrome patients
will yield the inhibitor genes that are associated with cellular senescence.
Another approach to this problem is to produce monoclonal antibodies
by immunizing mice with surface membrane preparations from senescent
cells. We have isolated monoclonal antibodies in this way.38 Of approximately
6000 hybridoma cultures screened, three yielded antibodies that reacted pref-
erentially with senescent cells but not with young cells. All the antibodies
react with an epitope on the fibronectin molecule. Although they react with
fibronectin from various sources, when it is denatured, they only react with
fibronectin distributed on the surface of senescent cells, not with fibronectin
distributed on the surface of young cells. This indicates that senescent cells
are producing either a fibronectin that is altered in its primary structure or
posttranslationally modified in a way different from that of young cells. The
senescent cell-produced fibronectin would then have a different conformation
than that of young cells. Another possibility is that the fibronectin protein
produced by senescent cells and young cells is the same, but the interaction
of fibronectin with other molecules produced by senescent cells and young
cells is different, thus leading to an altered confirmation of fibronectin as-
sociated with senescent cells, exposing an epitope which is sequestered in
young cells.

IV. DISCUSSION

The experimental results reviewed here indicate that cellular aging is an

active process, perhaps part of a genetic program, and that this genetic program
can be disrupted in various ways, giving rise to cellular immortality. We have
shown that there are at least four different ways that the normal cell processes
can be disrupted to lead to cellular immortality. One of the processes that
takes place in normal cells seems to be production of an inhibitor of the
initiation of DNA synthesis. This inhibitor is produced in quiescent cells, but
is reversible by the addition of growth factors or serum mitogens. The inhibitor
produced by senescent cells is not reversible. We do not know at the present
time whether the inhibitor produced by quiescent cells is the same as the
inhibitor produced by senescent cells. It is possible that they are the same,
but the control of expression of the inhibitors is different between young cells
and senescent cells. In senescent cells, the inhibitor is being produced con-
stitutively, whereas in young cells it is modulated in different parts of the
cell cycle and by growth factors.
Recently, changes in a number of cell cycle genes as cells become se-
nescent have been reported. These include failure to express c-fos or cdc2
when the cells are mitogenically stimulated,39'40 and the failure to phospho-
rylate the Rb gene.41 It may be that the defects in the regulation of these cell
cycle-related genes are responsible for senescent cells being unable to enter
into the S-phase. However, according to the data presented above, these
changes would be secondary to the expression of a cell surface inhibitor of
10 Transformation of Human Epithelial Cells

DNA synthesis by senescent cells. The mechanism by which this inhibitor

changes the expression of cell cycle genes remains to be elucidated. One
possibility is that the cell cycle genes are controlled by other events which
occur during the cell cycle, and since senescent cells are not cycling, these
genes are not triggered by the proper series of events.
The full significance of cellular aging in vivo is not known. However, it
may be that small decrements in various systems act synergistically. For
example, a decline in lung capacity coupled with a decline in cardiac output,
a decline in hemoglobin content, or the oxygen-carrying capacity of the blood
could lead to a significant decline in the total oxygen available. It is clear
that loss of cell proliferative capacity in some organ systems can have serious
consequences for the organism as a whole. For example, if the cells lining
the vascular system were not able to proliferate in response to injury, den-
udation of the vascular system could result. This could cause thrombosis or
atherosclerosis.
On the other hand, it seems likely that the limited proliferative potential
of normal cells in vivo is a powerful inhibitor of tumorigenesis. Even when
some of the changes leading to tumor formation have occurred, the limited
proliferative potential of nonimmortalized cells severely limits the damage
caused by these potentially tumorigenic cells.

REFERENCES
1. Liotta, L. A., Steeg, P. S., and Stetler-Stevenson, W. G., Cancer metastasis and
angiogenesis: an imbalance of positive and negative regulation, Cell, 64, 327, 1991.
2. Fearon, E. R. and Vogelstein, B., A genetic model for colorectal tumorigenesis, Cell,
61, 759, 1990.
3. O'Brien, W., Stenman, G., and Sager, R., Suppression of tumor growth by senescence
in virally transformed human fibroblasts, Proc. Natl. Acad. Sci. U.S.A., 1986, 83, 8659.
4. Hayflick, L. and Moorhead, P. S., The serial cultivation of human diploid cell strains,
Exp. Cell Res., 25, 585, 1961.
5. Hayflick, L., The limited in vitro lifetime of human diploid cell strains, Exp. Cell Res.,
37, 614, 1965.
6. Martin, G. M., Sprague, C. A., and Epstein, C. J., Replicative life-span of cultivated
human cells: effects of donor age, tissue, and genotype, Lab. Invest., 23, 86, 1970.
7. Norwood, T. H. and Smith, J. R., The cultured fibroblast-like cell as a model for the
study of aging, in Handbook of Biological Aging, Finch, C. E. and Schneider, E. L.,
Eds., Van Nostrand Reinhold, New York, 1985, 291.
8. Macieira-Coehlo, A., Implications of the reorganization of the cell genome for aging
or immortalization of dividing cells in vitro, Gerontology, 26, 276, 1980.
9. Meek, R. L., Bowman, P. D., and Daniel, C. W., Establishment of mouse embryo
cells in vitro. Relationship of DNA synthesis, senescence and malignant transformation,
Exp. Cell Res., 125, 453, 1977.
10. Rothfels, F. H., Kupelweiser, E. B., and Parker, R. C., Effects of x-irradiated feeder
layers on mitotic activity and development of aneuploidy in mouse embryo cells in vitro,
Can. Cancer Conf., 5, 191, 1963.
Smith 11

1 1 . Swim, H. E. and Parker, R. F., Culture characteristics of human fibroblasts propagated

serially, Am. J. Hyg., 66, 235, 1957.
12. Rohme, D., Evidence for a relationship between longevity of mammalian species and
life-spans of normal fibroblasts in vitro and erythrocytes in vivo, Proc. Natl. Acad. Sci.
U.S.A., 78, 5009, 1981.
13. Duthu, G. S., Braunschweiger, K. I., Pereira-Smith, O. M., Norwood, T. H., and
Smith, J. R., A long-lived human diploid fibroblast line for cellular aging studies:
applications in cell hybridization, Mech. Aging Dev., 20, 243, 1982.
14. Goldstein, S., Aging in vitro: growth of cultured cells from the Galapagos tortoise, Exp.
Cell Res., 83, 297, 1974.
15. Gorman, S. D., Hoffman, E., Nichols, W. W., and Cristofalo, V. J., Spontaneous
transformation of a cloned cell line of normal diploid bovine vascular endothelial cells,
In Vitro, 20, 339, 1984.
16. Littlefield, J. W., Attempted hybridization with senescent human fibroblasts, /. Cell
Physiol., 82, 129, 1973.
17. Pereira-Smith, O. M. and Smith, J. R., Expression of SV40 T antigen in finite life-
span hybrids of normal and SV40-transformed fibroblasts, Som. Cell Genet., 7, 411,
1981.
18. Pereira-Smith, O. M. and Smith, J. R., Evidence for the recessive nature of cellular
immortality, Science, 221, 964, 1983.
19. Pereira-Smith, O. M. and Smith, J. R., Genetic analysis of indefinite division in
human cells: identification of four complementation groups, Proc. Natl. Acad. Sci. U.S.A.,
85, 6042, 1988.
20. Ning, Y., Shay, J. W., Lovell, M., Taylor, L., Ledbetter, D. H., and Pereira-Smith,
O. M., Tumor suppression by chromosome 11 is not due to cellular senescence, Exp.
Cell Res., 192, 220, 1991.
21. Ning, Y. and Pereira-Smith, O. M., Molecular genetic approaches to the study of
cellular senescence, Mutn. Res., in press.
22. Ning, Y., Weber, J. L., Killary, A. M., Ledbetter, D. H., Smith, J. R., and Pereira-
Smith, O. M., Genetic analysis of indefinite division in human cells: evidence for a cell
senescence related gene(s) on human chromosome 4, Proc. Natl. Acad. Sci. U.S.A., 88,
5635, 1991.
23. Sugawara, O., Oshimura, M., Koi, M., Annab, L. A., and Barrett, J. C., Induction
of cellular senescence in immortalized cells by human chromosome 1, Science, 247, 707,
1990.
24. Norwood, T. H., Pendergrass, W. R., Sprague, C. A., and Martin, G. M., Dom-
inance of the senescent phenotype in heterokaryons between replicative and post-repli-
cative human fibroblast-like cells, Proc. Natl. Acad. Sci. U.S.A., 71, 2231, 1974.
25. Stein, G. H. and Yanishevsky, R. M., Entry into S phase is inhibited in two immortal
cell lines fused to senescent human diploid cells, Exp. Cell Res., 120, 155, 1979.
26. Norwood, T. H., Pendergrass, W. R., and Martin, G. M., Reinitiation of DNA
synthesis in senescent human fibroblasts upon fusion with cells of unlimited growth
potential, J. Cell Biol., 64, 551, 1975.
27. Stein, G. H., Yanishevsky, R. M., Gordon, L., and Beeson, M., Carcinogen-trans-
formed human cells are inhibited from entry into S phase by fusion to senescent cells
but cells transformed by DNA tumor viruses overcome the inhibition, Proc. Natl. Acad.
Sci. U.S.A., 79, 5287, 1982.
28. Yanishevsky, R. M. and Stein, G. H., Ongoing DNA synthesis continues in young
human diploid cells (HDC) fused to senescent HDC, but entry into S phase is inhibited,
Exp. Cell Res., 126, 469, 1980.
29. Drescher-Lincoln, C. K. and Smith, J. R., Inhibition of DNA synthesis in proliferating
human diploid fibroblasts by fusion with senescent cytoplasts, Exp. Cell Res., 144, 455,
1983.
30. Drescher-Lincoln, C. K. and Smith, J. R., Inhibition of DNA synthesis in senescent-
proliferating human cybrids is mediated by endogenous proteins, Exp. Cell Res., 153,
208, 1984.
12 Transformation of Human Epithelial Cells

31. Pereira-Smith, O. M., Fisher, S. F., and Smith, J. R., Senescent and quiescent cell
inhibitors of DNA synthesis. Membrane-associated proteins, Exp. Cell Res., 160, 297,
1985.
32. Stein, G. H. and Atkins, L., Membrane-associated inhibitor of DNA synthesis in
senescent human diploid fibroblasts: characterization and comparison to quiescent cell
inhibitor, Proc. Natl. Acad. Sci. U.S.A., 83, 9030, 1986.
33. Lumpkin, C. K. J., McClung, J. K., Pereira-Smith, O. M., and Smith, J. R.,
Existence of high abundance antiproliferative mRNA's in senescent human diploid fi-
broblasts, Science, 232, 393, 1986.
34. Lumpkin, C. K. J., McClung, J. K., and Smith, J. R., Entry into S phase is inhibited
in human fibroblasts by rat liver poly(A) + RNA, Exp. Cell Res., 160, 544, 1985.
35. Nuell, M. J., Stewart, D. A., Walker, L., Friedman, V., Wood, C. M., Owens,
G. A., Smith, J. R., Schneider, E. L., Dell'Orco, R., Lumpkin, C. K., Danner,
D. B., and McClung, J. K., Prohibitin, an evolutionary conserved intracellular protein
that blocks DNA synthesis in normal fibroblasts and HeLa cells, Mol. Cell. Biol., 11,
1372, 1991.
36. Pepperkok, R., Schneider, C., Philipson, L., and Ansorge, W., Single cell assay
with an automated capillary microinjection system, Exp. Cell Res., 178, 369, 1988.
37. Pepperkok, R., Zanetti, M., King, R., Delia, D., Ansorge, W., Philipson, L., and
Schneider, C., Automatic microinjection system facilitates detection of growth inhibitory
mRNA, Proc. Natl. Acad. Sci. U.S.A., 85, 6748, 1988.
38. Porter, M. B., Pereira-Smith, O. M., and Smith J. R., Novel monoclonal antibodies
identify antigenic determinants unique to cellular senescence, J. Cell Physiol., 142, 425,
1990.
39. Seshadri, T. and Campisi, J., Repression of c-fos transcription and an altered genetic
program in senescent human fibroblasts, Science, 247, 205, 1990.
40. Stein, G. H., Drullinger, L. F., Robetorye, R. S., Pereira-Smith, O. M., and Smith,
J. R., Senescent cells fail to express the cdc2 gene in response to mitogen stimulation,
submitted.
41. Stein, G. H., Beeson, M., and Gordon, L., Failure to phosphorylate the retinoblastoma
gene product in senescent human fibroblasts, Science, 249, 666, 1990.
Siegfried et al. 13

Chapter 2

DETECTION OF GROWTH FACTOR EFFECTS AND

EXPRESSION IN NORMAL AND NEOPLASTIC HUMAN
BRONCHIAL EPITHELIAL CELLS*

Jill M. Siegfried, Michael J. Birrer, and Frank C. Cuttitta

TABLE OF CONTENTS

I. Introduction 14

II. Purification of Growth Factors 14

III. Approaches to the Detection of Autocrine Growth Factors by

Epithelial Cells 15

IV. Effect of Known Peptides on Growth of Bronchial Epithelial

Cells 17

V. Effect of Known Peptides on Signal Transduction Pathways 18

VI. Culture of a Non-Small-Cell Lung Carcinoma in Growth

Factor-Free Medium 19

VII. Detection of Novel Growth Factor Activity 23

VIII. Conclusions 25

Acknowledgments 25

References 26

The opinions and assertions contained herein are the private views of the authors and are not
to be construed as official or reflecting the views of the Department of the Navy or the
Department of Defense.
14 Transformation of Human Epithelial Cells

I. INTRODUCTION

Both normal and neoplastic human epithelial and stromal cells have been
shown to produce peptides which are capable of stimulating the producing
cells themselves (autocrine growth factors1 4) or the surrounding cell types
which make up the tissue architecture in vivo (paracrine growth factors5'7).
While these factors may play a critical role in the growth and development
of normal tissues and organs, their local release in tumors may also be an
important factor in the uncontrolled growth of neoplasia. Normal cells have
been demonstrated to produce factors such as transforming growth factor a
(TGF-a) and insulin-like growth factor I (IGF-I) under conditions of cell
proliferation. 813 Thus, the basic mechanism of producing and responding to
growth factors through signal transduction in many cases may not be fun-
damentally different in normal and neoplastic cells. In fact, most of the
autocrine factors produced by tumors have some role in normal physiology,
although structural aberrations or mutations in growth factors or their receptors
are also known which are oncogenic.14'16 Alternate forms of growth factors
may also be produced by tumors,17 for which the role in normal physiology
is yet to be elucidated.
Neoplastic cells may therefore utilize existing mechanisms that bring
about cell proliferation to produce a microenvironment which supports con-
tinuous growth, while lacking responses to inhibitory mechanisms which
would control growth in normal tissues. Regardless of whether autocrine or
paracrine secretion is a cause or an effect of cell transformation, growth factors
or their receptors may be targets for new types of cancer therapy. 1820 A
knowledge of the biology of growth factors in normal and neoplastic human
cells is important in designing such therapeutic strategies. This chapter will
discuss some of the techniques which have been used by us and others to
detect growth factor production and secretion in human epithelial cells. We
will also discuss some of the specific growth factors which have been detected
in normal and neoplastic human epithelial cells in situ and in culture.

II. PURIFICATION OF GROWTH FACTORS

The classical approach for detection of secreted growth factors from a

tumor cell line was described by Marquardt and Todaro in 1982.21 These
investigators used biochemical methods to purify TGF-a from large volumes
of medium conditioned by sarcoma virus-transformed rat embryo fibroblasts
or a human melanoma cell line. TGF-a was shown to bind to the epidermal
growth factor (EGF) receptor and to stimulate anchorage-independent growth
of fibroblasts in consort with another secreted growth factor, transforming
growth factor (3, which was also present in conditioned medium.22 These
bioassays were used to monitor TGF-a activity during purification. Through
laborious work, they were able to purify 1.5 (Jig from 9.2 1 of medium
conditioned by melanoma cells. A human cDNA coding for TGF-a was
Siegfried et al. 15

subsequently isolated by Derynck et al. in 1984.23 TGF-a was shown to have

only 30 to 40% homology with EGF, but to fold into an almost identical
tertiary structure, allowing it to occupy the EGF receptor.24 TGF-a is produced
as a large prohormone which spans the cellular membrane; the mature 50-
amino acid peptide is probably cleaved by an elastase-like enzyme.25 This
transmembrane structure is shared by a family of proteins related to EGF
which have diverse functions, including growth factor activity, adhesion, and
protease activity.25
TGF-a has many functions in addition to stimulation of cell growth and,
for many, is more potent than EGF. For example, its effects on stimulating
cell motility, angiogenesis, and morphogenesis occur at lower concentrations
than EGF.25 It is expressed by normal cells during specific times in embryonic
development, and in adults can also be found in basal keratinocytes in skin8
and in proliferating mammary epithelial cells.11 It is found in many human
tumors and tumor cell lines and can be detected in the urine of patients with
disseminated cancer.26 In tumors, different high-molecular-weight forms of
TGF-a have been described, suggesting either alternate processing of the
prohormone or different levels of glycosylation.17-25
Another autocrine growth factor produced by many cell types is IGF-
j 3,12,13 IQP_I was first isolated from serum,27'29 but was later found to be
produced by the liver30-31 as well as other tissues,32 and to be locally released
in situ33 and in culture.34"36 It appears to be an important autocrine factor in
cancer of the lung3'37'38 and breast.39 41 Insulin-like growth factor II (IGF-II),
which has a structure similar to that of IGF-I, appears to only be released
normally in embryonic tissues.28>32-42 It also may be an important autocrine/
paracrine growth factor in human tumors.43 Basic fibroblast growth factor
(bFGF) or the related int-2 gene product has also recently been shown to be
expressed by some prostate tumors,44 and to stimulate both mesenchymal45
and some epithelial cells46 in culture. Since bFGF is one of the major factors
responsible for the growth-promoting effects of bovine pituitary extract (BPE)
toward epithelial cells,47 it is also a candidate autocrine/paracrine factor for
carcinomas. The gastrin-releasing peptide (GRP)/bombesin family has been
shown to act as an autocrine factor in small-cell carcinoma,4 but since fibro-
blasts also respond to these neuropeptides, the paracrine effects of bombesin-
like peptides may also be important in tumor growth. Another candidate
paracrine factor which transformed epithelial cells may secrete is platelet-
derived growth factor (PDGF). PDGF is highly growth-stimulatory for mes-
enchymal cells,48 and may be produced by some carcinomas.6

III. APPROACHES TO THE DETECTION OF AUTOCRINE

GROWTH FACTORS BY EPITHELIAL CELLS

A complication in applying large-scale biochemical purification to the

identification of secreted growth factors from primary epithelial tissues or
cultured epithelial cells is that many tumors, as well as normal tissues, produce
16 Transformation of Human Epithelial Cells

a mixture of factors which may include inhibitory as well as stimulatory

peptides. The question becomes how to separate and identify each of these,
determine if any are unique, and demonstrate a cellular response to them,
using very small amounts of material. How, then, to further establish an
autocrine or paracrine role for these factors?
We have approached this problem in the study of normal and neoplastic
human bronchial epithelial cells in a number of different ways. First, we have
used primary human bronchial epithelial cells (HBE cells) and primary and
secondary cultured non-small-cell lung carcinoma cells (NSCLC cells) in
colony assays to screen known peptides for the ability to stimulate growth.
We have examined peptides shown to be autocrine growth factors for small-
cell lung cancer and for carcinomas derived from other tissues, as well as
ectopic peptides known to be expressed clinically in non-small-cell lung tu-
mors. Second, we have examined the ability of known peptides to activate
signal transduction pathways in normal and neoplastic lung epithelial cells.
These two approaches are intended to identify peptide hormones which are
mitogens for lung cells and are therefore candidate autocrine factors which
might also be produced by lung tissues. Third, we have adapted an NSCLC
cell line to grow in completely serum-free conditions, without any exogenous
growth factors, and have used medium conditioned by these cells as a source
of growth factor activity. This approach creates conditions in which NSCLC
cells are forced to produce growth factors which are needed for their own
proliferation. Media conditioned by tumor cells under these conditions can
be concentrated and used as a source of autocrine or paracrine factors. We
have assayed both crude extracts and fractions separated by high-pressure
liquid chromatography for growth-stimulating activity and for the presence
of known mitogenic peptides. We have also used monoclonal antibodies raised
against growth factor receptors to block effects of conditioned medium in
order to demonstrate that the peptides detected are responsible for the observed
growth stimulation.
We have also applied our in vitro system for detecting proliferation of
HBE cells and lung tumor cells in response to novel growth factors. For
example, we have examined the known sequences of peptide prohormones
for motifs downstream of the mature peptides that are indicative of protein-
processing enzymes. The presence of such motifs implies that the sequence
found within the motif is a potential new growth factor, released during
processing of the prohormone. We have synthesized putative peptide growth
factors based on such sequences and have been able to demonstrate that they
have biological effects in our system.49-50 Below we will illustrate how each
of these approaches has been useful in defining growth factors important in
the proliferation of normal and neoplastic bronchial epithelial cells. As new
growth factors are discovered, all of these approaches can be used to determine
if they have a role in the growth of cells derived from the lung or other
tissues.
Other documents randomly have
different content
 DISCOVERY OF OCEAN COUNTY. S" heen " Elected for
Shrewsbury " as a Deputy, but liis place declared vacant, probably
because he had been selected by the Governor as a member of the
council at that time. Colonel Morris had purchased a large tract of
land, in what was afterwards known as Monmouth County, October
25tli, 1676, said to contain 3,540 acres, whereu23on he located, as
described in 1680, " his iron mills, his Manors, and divers other
buildings for his servants and dependants ; together with 60 or 70
negroes about the Mill and Husbandry. To this plantation he gave the
name of Tintern (corrupted afterwards to Tinton) after an estate
which had belonged to the family in Monmouthshire, England, and
from him Monmouth county received its name." DISCOVEEY OF
OCEAN COUNTY. Who first discovered this section of our country?
Who first entered Barnegat Bay, and explored its shores ? Who were
the first whites who located here ? Have any accounts of the Indians
once living here been preserved ? These are among the first
questions which naturally present themselves in making inquires into
the early history of this section of our State. While the records of the
past, meagre indeed as regards this locality, do not furnish as full
answers as desirable, yet much has been preserved which is of
interest to all desirous of obtaining information on these and kindred
points. The discovery of that part of New Jersey now known as
Ocean County, was by Sir Henry Hudson, on the 2d day of
September, 1609, while cruising along our coast in the celebrated
Dutch ship, the Half Moon. This ship was quite small, being of only
eighty tons burthen, and of a build that would now be considered
quite novel, reminding one of the curious-looking Dutch galliots,
which occasionally were seen in the harbor of New York a generation
or so ago, which used to attract the attention of, and are well
remembered by old seafaring men of Ocean County.
 38 HISTORY or MONMOUTH AND OCEAN COUNTIES. This
ship, two or three days previously, had tried toenter Delaware Bay,
but finding the navigation dangerous, no attemjDt was made to
land, and she again stood out to sea. After getting fairly out, Hudson
headed north-eastwardly, and after a while hauled in and made land,
Sept. 2d, near Egg Harbor. A very complete log^ of the ship was
kept by the mate, Alfred Juet, and the part relating to Monmouth
and Ocean counties is published elsewhere in these pages. Samuel
H. Shreve, Esq., who in past years has furnished many valuable
historical items to the New Jersey Courier ^ says in a
communication dated January, 1868 : " Ferrago Forge was erected
by Gen. Lacey in 1809, and the same year Dover Forge was built by
W. L. Smith,, the son-in-law of Lacey." It has been stated that Lacey
expended ten thousand dollars at Ferrago in building the dam alone,
and the construction of the forge and other buildings and of the road
to Forked River must have required a very considerable outlay of
money. OLD MONMOUTH DESCRIBED BY AN ANCIENT WRITER.
MIDDLETOWN, SHREWSBURY AND FREEHOLD IN 1708. ^sK^
JERSEY A PARADISE. We copy the following from the celebrated but
quite rare work of Oldmixon, published in 1708. The capitals,
orthography and italics are about as in the original. After describing
Middlesex county, he says : " We cross over the river from Middlesex
into Montnouth County ; Where w^e first meet with 3Hddleton a
pretty Good Town consisting of 100 Families and 30,000 Acres of
Ground on what they call here Out Plantations. 'Tis about 10 or 12
miles over Land, to the Northward of Shrewsbury and 26 miles to
the Southward of Piscattaway. Not far off, the Shoar winds itself
about like a Hook and being sand}^ gives Name to all the Bay.
Shrewshury is the most Southern Town of the Prov 
 OLD MONMOUTH DESCEIBED BY AN ANCIENT WRITER. 39
ince and reckon'd the cliief Towu of tlie Shire. It contains about 160
Families and 30,000 Acres of ^/?/?' Plantations, belonging to its
Division. 'Tis situated on the Side of a fresh Water Stream, thence
called Shrewsbury Riyer, not far from its Mouth. Between this Town
and Middleton is an Iron Work but we do not understand it has been
any great Benefit to the Proprietors. Cob Morris is building a Church
at the Falls. There's a new town in the County called Freehold, which
has not been laid out and inhabited long. It does not contain as yet
above 40 Families and as to its Oiit Plantations we suppose they are
much the same in number with the rest and may count it about
30,000 acres. We have not divided the counties into Parishes and
that for a good reason, there being none, nor indeed a Church in the
wdiole Province Avortli that Name. But there are several
Congregations of Church of England men as at Shreivslniry, Amh.y,
Elizaheth Town SiVi^ Freehold whose Minister is Mr. John Beak ; his
Income is 65^ a year ; and a Church is building at Salem. In another
place Oldmixon in speaking of the first settlers of New Jersey saj-s :
" We must note that most of the first English Inhabitants in this
country (East and West Jersey) were Dessenters, and most of them
Quakers and Anabajjtists. These people are generally industrious ;
Be their Hypocrisy to themselves if they are Hypocrites ; but we
must do them the Justice to own that they are the fittest to inhabit a
new discovered Country, as possessing Industry, and shunning those
public Vices which beget Idleness and Want. Their enemies drove
great numbers of them out of England, and the Jerseys had their
share of them. The People here are for this Beason Dissenters to this
Day, there being but two Church of England Ministers in both
Provinces ; and this may be one reason why there are no Parish
Churches, which the Inhabitants may be afraid to build, least it
might be a temptation for more Orthodox Di^dnes to come amona:
them.
 40 HISTORY OF MONMOUTH AND OCEAN COUNTIES. "A
gentleman asking one of tlie Proprietaries '■If tit ere were no
Lwv^yers in the Jerseys?' "Was answered ' JVo.'' And then ' If there
were no Physicians f ' The Proprietor replied ' Wo.' ' Nor Parsons f '
adds the Gentleman. ' iV^c,' says the Proprietor. Upon which the
other crj'd ' What a happy place must this he and hov:) ivorthy the
name of Paradice ! ' We do not perhaps differ more from this
gentleman than we agree with him." Oldmixon derived his
information of New Jersey from two of the Proprietors as will be
seen by the following extract from his preface : " Mr. Docliv^ra and
Dr. Cox were both so kind as to inform him fully of the Jeeseys and
Mr. Pen did him the same Favor for Pennsylvania ; these three
Gentlemen doing him the Honor to admit him into their Friendship."
OLD MONMOUTH UNDER THE DUTCH. Ex-Governor Parker, dec'd, in
his valuable address before the Neiv Jersey Historical Societ}-,
produced the old town book of Middletown township, which gives
the history of this section of East Jerse}- from 1667 to 1702. After
the Dutch conquest in 1673, it was stated that little or nothing is
recorded in the town book during their brief rule of less than a year.
The Dutch had the sujjremacy in New York and New Jersey until
1664, when the English conquered the Dutch. In 1673, a war having
again broken out between England and Holland, a small Dutch
squadron was sent over and arrived at Staten Island, July 30th.
Captain Manning, the English officer temporarily in command at New
York, surrendered at once without any effort to defend the place and
the Dutch again resumed sway over J^ew York, New Jersey and
settlements along the Delaware. They retained it however only a few
months, as by a treaty made in February following, these places
were ceded back to England, though the English appear not to have
taken formal possession until November fol 
 OLD MONMOUTH UNDER THE DUTCH. . 41 lowing. Dui'iug
this sliort time while the Dutch were again in authority, embracing
the time that the Midclletowu township book records but little or
nothing, the following items relating to Old Monmouth, are found
among the official records of the Dutch at New York. The first is an
order issued shortly after their arrival ; the ortho■grajDliy is given as
found. " The inhabitants of Middletowu and Shrewsbury, are hereby
charged and required to send their deputies unto us on Tuesday
morning next, for to treat with us upon articles of surrendering their
said towns under the obedience of their High and Mighty Lords, the
States General of the said United Provinces, and his serene
Highness, the Prince of Orange, or by refusall we shall be
necessitated to subdue the places thereunto by force of arms. "
Dated at New Orange this 12tli day of August, A. D. 1673. " coenelis
evertse, jr. "Jacob Benckes." In compliance with the above order,
deputies from Shrewsbury, Middletowu and other places in East
Jersey, appeared in court on the 18th of August, and upon their
verbal request the same privileges were granted to them as to Dutch
citizens. " August 19th, 1673. Middletowu, Shrewsbury and other
towns in Achter Coll, to name two deputies each, who shall
nominate three persons for Schout and three for Secretarys, out of
which said nominated persons by us shall be elected for each town,
three magestrates and for the six towns, one Schout, and one
Secretary. "Jacob Benckes. " CoRNELIS EVERTSE, Jr." Achter Coll
above mentioned, is said to mean " beyond the hills," that is, beyond
Bergen Hills. The Dutch in New York, it is stated, sometimes called
Old Monmouth and other parts of East Jersey, beyond Bergen Hills,
by this name. " April lOfch, 1671. A certain proclamation being de 
 42 HISTORY OF MONMOUTH AND OCEAN COUNTIES.
liverecl into Couucil from the Magestrates of the Tonne of
Middletoune, prohibiting all inhabitants from departing out of said
tonne, unless they give bail to return as soon as their business will
have been performed, or they be employed in public service &c.,
requesting the Governers approval of the same, which being read
and considered, it is resolved and ordered by the Governer General
and Council, that no inhabitant can be hindered changing his
domicile, within the Pro^dnce unless arrested for lawful cause ;
however ordered that no one shall depart from the tonne of
Middletoune, unless he previously notifies the Magestrates of his
intention." CAUSES OF THE KEVOLUTION - PRINCIPLES
INVOLA^ED. EARLY STAND TAKEN BY THE CITIZENS OF
MONMOUTH. — PROCEEDINGS OF MEETINGS IN DIFFERENT
TOWNSHIPS IN 1774-5. — FREEHOLD LEADS THE STATE. —
COUNTY RESOLUTIONS.— AN ADMIRABLE DOCUMENT. —
PATRIOTS APPEAL TO THEIR DESCENDANTS. — "A FAITHFUL
RECORD" OF 1774. Historians of other States have always conceded
that the citizens of New Jersey were among the earliest and most
active opponents of those tyrannical acts of Great Britain which
brought on the w^ar, and finally resulted in separation. Large and
spirited public meetings were held in various parts of the State in
1774-5, to denounce the obnoxious laws, and to organize for
counsel and defence. At this stage of affairs, separation from
England had not been proposed, and most of these meetings, while
condemning the acts of the British Ministry and Parliament, still
expressed decided loyalty to the King. Our ancestors warmly
seconded the stand taken by the people of Boston, and freely
forwarded contributions to the suffering inhabitants of that city. We
annex extracts from the proceedings of some of these meetings in
Old Mcmmouth, as they exhibit the timely zeal and firm and decided
spirit of its citizens, and
 CAUSES OF THE REVOLUTION — PRINCIPLES INVOLVED.
43 also furuisli the names of some of tlie leading- spirits who were
prominent in the early stages of political movements which brought
on the Revolution. The several counties of the State were requested
to send delegates to meet at New Brunswick, July 21st, 1774, to
consider what action should be taken by the citizens of the province
of New Jersey. .This convention was generally spoken of as the
"Provincial Congress of New Jersey," and was a different body from
the Legislature ; in several instances, however, the same persons
were members of both bodies. A number of persons named in these
proceedings were afterwards, during the war, conspicuous in military
or civil life, for their services in behalf of their country in legislative
halls and on the field of battle. For a year or two the citizens of the
county appeal to have been about unanimous in their sentiments,
but when finally the subject of a separation from the mother country
was boldly advocated, there was found to be a diversity of opinion,
and some who were among the most active in the meetings of 1774-
5, earnestly opposed the proposition, and eventually sided wdth
England in the later years of that memorable struggle. The fearful
consequences of this division, in which it would seem almost every
man capable of bearing arms was compelled to take sides, we have
endeavored to give in other chapters. The citizens of Freehold had
the honor, we believe, of holding the first meeting in New Jersey to
denounce the tyrannical acts of Great Britain — of inaugurating the
movements in our State which finally resulted in Independence. The
date of their first meeting is June 6th, 1774 ; the earliest date of a
meeting in any other place that we have met wdth, is of a meeting
at Newark, June 11th, 1774. The following is a copy of the Freehold
Proceedings : Lower Freehold Resolutions. "Freehold June 6tli 1774.
"At a meeting of the Freeholders and Inhabitants of the Township of
Lower Freehold in the county of
 44 HISTORY OF MONMOUTH AND OCEAN COUNTIES.
Moiimontli in New Jersey, on Monday the 6tli day of June, 1774,
after notice given of tlie time, place and occasion of this meeting : "
Besolved That it is the unanimous opinion of this meeting, that the
cause in which the inhabitants of the town of Boston are now
suffering is the common cause of the whole Continent of North
America ; and that unless some general spirited measures, for the
public safety be speedily entered into there is just reason to fear
that every Province may in turn share the same fate with them ; and
that therefore, it is highly incumbent on them all to unite in some
effectual means to obtain a repeal of the Boston Port Bill and any
other that may follow it, which shall be deemed subversive of the
rights and privileges of free born Americans. "And that it is the
opinion of this meeting that in case it shall hereafter appear to be
consistent with the general opinion of the trading towns and the
commercial part of our countrymen, that an entire stoppage of
importation and exportation from and to Great Britain and the West
Indies, until the said Port Bill and other Acts be repealed, will be
conducive to the safety and preservation of North America and her
liberties, they will yield a cheerful acquiescence in the measure and
earnestly reccommend the same to all their brethren in this
Province. " Resolced, vioreover. That the inhabitants of this township
will join in an Association with the several towns in the county and in
conjunction with them, with the several coiinties in the Province (if,
as we doubt not they see fit to accede to the proposal) in any
measures that may appear best adapted to the weal and safety of
North America and all her loyal sons. " Oi'dered That John Anderson
Esq Peter Forman Hendrick Smock John Forman AsHER Holmes
Capt. Jno. Covenhoven and Dr. Nathaniel Scudder be a committee
for the township to join those who may
 CAUSES OF THE REVOLUTION — PRINCirLES INVOLVED.
45 be elected for tlie ueigliboring townships or counties to constitute
a General Committee for any purposes similar to those above
mentioned; and that the gentlemen so appointed do immediately
solicit a correspondence with the adjacent towns." (Dr. Scudder
subsequently was a Colonel in the First Regiment Monmouth Militia,
and killed October 15th, 1781, as described elsewhere.) The
following week the citizens of Essex sent the following to the patriots
of Monmouth : Essex to Monmouth. " Elizabethtown June 13 1774
"Gentlemen: The alarming Measures which have been lately taken to
deprive the Inhabitants of the American Colonies of their
constitutional Rights and Privileges, together with the late violent
attacks made upon the rights and liberties of the Colony of the
Massachusetts Bay (for asserting and endeavoring to maintain their
rights) manifestly intended to crush them without Mercy and thereby
disunite and weaken the Colonies, and at the same time dare them
to assert or own their Constitutional Rights, Liberties or Pro23erties,
under the Penalty of the like, and if possible, worse treatment : and
as the Assembly of New Jersey are not like to meet in time to
answer the Design proposed, and the neighboring Colonies are
devising and expecting the immediate union of this Colony with
them. "Sundry of the Inhabitants of the County of Essex by
Advertisements, convened a general Meeting of said County at
Newark on Saturday last, when the said inhabitants unanimously
entered into certain Resolves and Declarations upon that occasion, a
copy of which you have enclosed. We the Committee appointed by
the said Meeting, do earnestly request that You will immediately by
Advertisements or otherwise, call a general Meeting of your County
for the purposes aforesaid as soon as possible, as we have
intelligence that it is most probable the General Congress of the
Colonies will be held the latter end of July next. We think New"
Brunswick the most
 46 HISTORY OF MONMOUTH AND OCEAN COUNTIES.
suitable place for tlie committee to meet, and with submission to
tliem desire they will meet us at New Bruuswick on Thursday, July
21st next, at 10 o'clock in the morning, unless some other time and
place more suitable shall in the meantime ])e agreed upon. "We
earnestly request your answer as soon as possible. "Letters of this
Tenor and Date we now despatch to the other Counties in this
Colony. We are, Gentlemen, "your most ob't servants Stephen Crane,
Chairman. "By order; "To Messrs. Edward Taylor, Richard Lawrence,
Elisha Lawrence, John Taylor and Henry Waddell and other
Inhabitants of the County of Monmouth, Friends to the Liberties and
Privileges of the American Colonies." (The above letter was directed
to the above named gentlemen "or to any body else in Monmouth
County.") Delegates from the different townships in the county
assembled at Freehold, July 19th, and the result of their decision is
found in the following admirable document. It is lengthy, but will
well repaj^ perusal. In the closingparagraph they trust that some
faithful record will transmit the reasons Avhich actuated them, to
their posterity to whom they make a brief but eloquent appeal. As
they desired, this record has been preserved, and as they desired,
we do what we can to place it before their descendants : Monmouth
County Eesolutions. " On Tuesday, July 19th, 1774, a majority of the
Committees from the several townships in the County of Monmouth
of the Colony of New Jersey, met according to appointment at the
Court House at Freehold in said county ; and appearing to have
been regularly chosen and constituted by their respective townships,
they unanimously agreed upon the propriety and expediency of
electing a committee to represent the whole county at the
approaching Provincial Convention to be held at the
 CAUSES OF THE REVOLUTION — nUNCirLES INVOLVED. 47
city of New Brunswick, for the necessary purpose of constituting
delegates from this Province to the general Congress of the Colonies
and for all other such important purposes as shall hereafter be found
necessary. " They at the same time also recorded the following
Resolutions, Determinations and Opinions, which they " wish to be
transmitted to posterity as an ample testimony to their loyalty to his
British Majesty, of their firm attachment to the principles of the
glorious Revolution and their fixed and unalterable purpose, by every
lawful means in their power, to maintain and defend themselves in
the possession and enjoyment of those inestimable civil and religious
privileges which their forefathers, at the expense of so mncli blood
and treasure, have established and handed down to them. "1st. In
the names and behalf of their constituents, the good and loyal
inhabitants of the county of Monmouth, in the colony of New Jersey,
they do cheerfully and publicly proclaim their unshaken allegiance to
the person and government of his most gracious Majesty, King
George the Third, now on the British throne, and do acknowledge
themselves bound at all times, and to the utmost exertion of their
power to maintain his dignity and lawful sovereignty in and over all
his colonies in America ; and that it is their most fervent desire and
constant prayer that in a Protestant succession, the descendants of
the illustrious House of Hanover, may continue to sway the British
sceptre to the latest posterity. " 2d. They do highly esteem and prize
the happiness of being governed and having their liberty and
property secured to them by so excellent a system of laws as that of
Great Britain, the best doubtless in the universe ; and they will at all
times cheerfully obey and render every degree of assistance in their
power to the full and just execution of them. But at the same time
will, with the greatest alacrity and resolution oppose any
umvarrantable innovations in them or any additions to or alterations
in the grand system which may appear unconstitutional, and
consequently inct^nsistent with the
 48 HISTOEY OF MONMOUTH AND OCEAN COUNTIES.
liberties and privileges of the descendants of free born American
Britons. " 3d. As there has been for ages past, a most happy union
and uninterrupted connection between Great Britain and her colonies
in America, they conceive their interests are now become so
intimately blended together and their mutual dependence upon each
other to be at this time so delicately great that they esteem
everything wdiich has a tendency to alienate affection or disunite
them in any degree, highly injurious to their common happiness and
directly calculated to produce a Bevolution, likely in the end to
proA'e destructive to both ; they do therefore heartily disclaim every
idea of that spirit of independence which has, of late, by some of our
mistaken brethren on each side of the Atlantic, been so groundlessly
and injuriously held up to the attention of the nation, as having
through ambition, possessed the breasts of the Americans. And
moreover they do devoutlj' beseech the Supreme Disposer of all
events, graciously to incline the heart of our Sovereign and all his
Ministers, to a kind and impartial investigation of the real sentiments
and disposition of his truly loyal American subjects. "4th.
Notwithstanding many great men and able writers have employed
their talents and pens in favor of the newly adopted mode of
taxation in America, they are yet sensible of no convictive light being
thrown upon the subject ; and therefore, although so august a body
as that of the British Parliament is now actually' endeavoring to
enforce in a military way, the execution of some distressing edicts
upon the capital of the Massachusetts colony, they do freely and
solemnly declare that in conscience they deem them, and all others
that are, or ever may be framed upon the same principles,
altogether unprecedented and unconstitutional, utterly inconsistent
with the true original intention of Magna Charta, subversive of the
just rights of free born Englishmen, agreeable and satisfactory only
to the domestic and foreign enemies of our nation, and consequently
pregnant with complicated
 CAUSES OF THE REVOLUTION — PRINCIPLES INVOLVED.
49 ruin, aud tending directly to the dissolution and destruction of the
British Empire. " 5th. As they, on the one hand firmly believe that
the inhabitants of the Massachusetts colony in general, and those of
the town of Boston in particular, are to all intents and purposes as
loyal subjects as any in all his "Majesty's widely extended dominions
; and on the other, that (although the present coercive and
oppressive measures against them may have taken rise in some part
from the grossest and most cruel misrepresentation both of their
disposition aud conduct) the blockade of that town is principally
designed to lead the way in an attempt to execute a dreadful deep
laid plan for enslaving all America. They are therefore clearly of
opinion, that the Bostouians are now eminently suffering in the
common cause of American freedom, and that their fate may
probably prove decisive to this very extensive continent and even to
the whole British nation ; and they do verily expect that unless some
generous spirited measures for the public safety be speedily entered
into aud steadily prosecuted, every other colony will soon in turn feel
the pernicious effects of the same detestable restrictions. Whence
they earnestly entreat every rank, denomination, society and
profession of their brethren, that, laying aside all bigotry and every
party disposition, they do now universally concur in one generous
and vigorous effort- for the encouragement and support of their
suffering friends, and in a resolute assertion of their birthright,
liberties and privileges. In consequence of which they may
reasonably expect a speedy repeal of all the arbitrary edicts
respecting the Massachusetts government, and at the same time an
effectual preclusion of any future attempts of the kind from the
enemies of our happy Constitution, either upon them or any of their
American brethren. " 6th. In case it shall hereafter appear to be
consistent with the result of the deliberation of the general
Congress, that an interruption or entire cessation of commercial
intercourse with Great Britain and even
 50 HISTORY OF MONMOUTH AND OCEAN COUNTIES.
(painful as it may be) with the West Indies, until such oppressive
Acts be repealed and the liberties of America fully restored, stated
and asserted, will on this deplorable emergency be really necessary
and conducive to the jjublic good, they promise a ready
acquiescence in every measure and will recommend the same as far
as their influence extends. " 7th. As a general Congress of Deputies
from the several American Colonies is proposed to be held at
Philadelphia soon in September next, they declare their entire
approbation of the design and think it is the only rational method of
evading those aggravated evils which threaten to involve the whole
continent in one general calamitous catastrophe. They are therefore
met this day, vested with due authority from their respective
constituents, to elect a committee to represent this county of
Monmouth in any future necessary transactions respecting the cause
of liberty and especially to join the Provincial Convention soon to be
held at New Brunswick, for the purpose of nominating and
constituting a number of Delegates, who in behalf of this Colony
may steadily attend to said general Congress and faithfull}^ serve
the laboring cause of freedom and they have consequently chosen
and deputed the following gentlemen to that important trust viz :
Edward Taylor John Anderson John Taylor Dr. Nathaniel Scudder
John Burro wes John Covenhoven Joseph Holmes Josiah Holmes
Edward Williams James Grover John Lawrence. "Edward Taylor being
constituted chairman and any five of them a suflicient number to
transact business. And they do beseech, entreat, instruct and enjoin
them to give their voice at said Provincial Convention, for no persons
but such as they in good conscience and from the best information
shall verilj^ believe to be amply qualified for so interesting a
department ; particularly that they be men highly approved for
integrity, honesty
 CAUSES OF THE DEVOLUTION — PEINCirLES INVOLVED.
51 and uprio-lituess, faithfully attaclied to liis Majesty's person and
lawful government, well skilled in the principles of our excellent
constitution and steady assertors of all our civil and religious
liberties. "8th. As under the present operation of the Boston Port Bill,
thousands of our respscted brethren in that town must necessarily
be reduced to great distress, they feel themselves affected with the
sincerest sympathy and most cordial commiseration ; and as they
expect, under God, that the final deliverance of America will be
owing;, in a great degree, to a contiuuancs of their virtuous
struggle, they esteem themselves bound in duty and in interest to
afford them every assistance and alleviation in their power ; and
they do now in belief of their constituents, declare their readiness to
contribute to the relief of the suffering poor in that town ; therefore
they request the several committees of the country, when met, to
take into serious consideration the necessitj^ and expediency of
forwarding under a sanction from them, subscriptions through every
part of the Colony, for that truly humane and laudable purpose ; aud
that a proper plan be concerted for laying out the i3roduct of such
subscriptions to the best advantage, and afterwards transmitting it
to Boston in the safest and least expensive way. " 9th. As we are
now by our Committees in this, dn conjunction with those of other
colonies, about to delegate to a number of our countrymen a power
equal to any wherewith human nature alone was ever invested ; and
as we firmly resolve to acquiesce in their deliberations, we do
therefore earnestly entreat them, seriously and conscientiously to
weigh the inexpressible importance of their arduous department,
and fervently to solicit that direction and assistance in the discharge
of their trust, which all the powers of humanity cannot afford them ;
and we do humbly and earnestly beseech that God, in whose hand
are the hearts of all flesh and who ruleth them at his pleasure,
graciously to infuse into the whole Congress a spirit of true wisdom,
prudence and
 52 HISTORY OF MONMOUTH AND OCEAN COUNTIES. just
moderation ; and to direct them to sucli unanimous and liappy
conclusion as shall terminate in His own honor and glory, the
establishment of the Protestant succession of the illustrious House of
Hanover, the mutual weal and advantage of Great Britain and all her
Dominions and a just and permanent confirmation of all the civil and
religious liberties of America. And now lastly, under the consideration
of the bare possibility that the enemies of our constitution will yet
succeed in a desperate triumph over us in this age, we do earnestly
(should this prove the case) call upon all future generations to renew
the glorious struggle for liberty as often as Heaven shall afford them
any probable means of success. " May this notification, by some
faithful record, be handed down to the yet unborn descendants of
Americans, that nothing but the most fatal necessity could have
wrested the present inestimable enjoyments from their ancestors.
Let them universally inculcate upon their beloved offspring an
investigation of those truths, respecting both civil and religious
liberty, which have been so clearly and fully stated in this generation.
May they be carefully taught in all their schools ; and may they
never rest until, through Divine blessing upon their efforts, true
freedom and liberty shall reign triumphant over the whole Globe. "
Signed by order of the Committees, " Edwaed Tayloe Chairman."
BOSTON GEATEFULLY ACKNOWLEDGES MONMOUTH
CONTRIBUTIONS. The patriots of Monmouth promptly and freely
contributed to the suffering inhabitants of Boston. In forwarding
their first contribution " they entreated their brethren not to give up,
and if they should want a further supply of bread to let them know
it." On the 21st of October, 1774, a letter was written on behalf of
the Bostonians, to the citizens of Monmouth, in which they say :
 BOSTON ACKNOWLEDGES MONMOUTH CONTEIBUTIONS.
53 "The kind and generous donations of tlie County of Monmoutli in
the Jersies we are now to acknowledge and with grateful hearts to
thank you therefor, having received from the Committee of said
county, per Captain Brown, eleven hundred and forty (1140) bushels
of rye and fifty barrels of rye meal, for the suffering poor of this
town, which shall be applied to the purpose intended by the donors ;
and what further cheers our hearts, is your kind assurances of a
further supply, if necessary, to enable us to oppose the cruel
Parliamentary Acts, levelled not only agaius't this town, but our
whole Constitution." "Committees of Observation and Inspection."
"Freehold December 10th 1774 "In pursuance of the
recommendation of the Continental Congress and for the
preservation of American Freedom, a respectable body of the
freeholders of Freehold township met at the Court House and
unanimously elected the following gentlemen to act as a Committee
of Observation and Inspection for said township : John Anderson
Hendrick Smock John Forman John Covenhoven Asher Holmes Dr.
Nath'l Scudder Peter Forman David Forman Dr. T. Henderson. "The
committee were instructed by their constituents" to carry into
execution the several important and salutary measures pointed out
to them by the Continental Congress and without favor or affection
to make all such diligent inquiry as shall be found conducive to the
accomplishment of the great necessary purposes held up to the
attention of Americans." Upper Freehold, Dover and Middletown
formed similar committees, and notified the Freehold committee.
Shrewsbury however failed to appoint a committee. This may have
been owing to the prevalence of Quaker principles in the township.
An attempt by the patriots of Shrewsbury was made to have a
Committee appointed,
 54 HISTORY OF MONMOUTH AND OCEAN COUNTIES. as
will be seen by tlie following copy of an advertisement put up in this
township : " Adyehtisement. " Shrewsbury January 2nd 1775.
"Agreeable to the Resolutions of the late General Continental
Congress — The Inhabitants of the town of Shrewsbury, more
especially such as are properly qualified for choosing
Representatives to serve in the General Assembly are hereby warned
to meet at the house of Josiah Halstead, in said Shrewsbury, on
Tuesday the 17tli of this instant January at noon, in order to choose
a Committee for the several purposes as directed by the said
Congress. "As the method ordered by the Congress seems to be the
only peaceable method the case will admit of, on failure of which
either comfirmed Slavery or a civil war of course succeeds ; the bare
mention of either of the two last is shocking to human nature, more
particularly so to all true friends of the English Constitution.
"Therefore it becomes the indispensable duty of all such to use their
utmost endeavors in favor of the first or peaceable method, and
suffer it not to miscarry or fail of its salutary and much desired
effects by means of any sinister views or indolence of theirs. Surely
expecting on the one hand to be loaded with the curses arising from
slavery to the latest jjosterity, or on the other hand the guilt of blood
of thousands of their brethren and fellow Christians to lay at their
door and to be justly required at their hands. " Think well of this
before it be too late and let not the precious moments pass." A
number of the citizens of Shrewsbury assembled at the time and
place mentioned in the advertisement but they failed to appoint a
committee. The following shows the conclusion to which the meeting
came. It concludes more like a Quaker Meeting epistle than a town
meeting resolve : "Extract from a letter to a gentleman in New York
dated Shrewsbury N. J. January 18tli 1775.
 BOSTON ACKNOWLEDGES MONMOUTH CONTRIBUTIONS.
55 "111 consequence of an anonymous advertisement fixed up in
this place, gi^'ing notice to freeholders and others, to meet on
Tuesday the 17th inst. in order to choose a Committee of Inspection,
etc., between thirty and forty of the most respectable freeholders
accordingly met and after a few debates on the business of the day,
which were carried on with great decency and moderation it was
generally agreed (there being only four or five dissenting votes) that
the appointment of a committee was not only useless, but they were
apprehensive would prove a means of disturbing the peace and
quietness which had hitherto existed in the township, and would
continue to use their utmost endeavors to preserve and to guard
against running upon that rock on which, with much concern, they
beheld others, through an inattentive rashness, daily splitting." The
Freehold Committee of Observation and Inspection at a meeting
held March 17tli, 1775, took up the case of Shrewsbury township,
and after stating the subject in a preamble they resolved that from
and after that day they would esteem and treat the citizens of
Shrewsbury as enemies to their King and country and deserters of
the common cause of Freedom ; and would break off all dealings
and connections with them "unless they shall turn from the evil of
their ways and testify their repentance by adopting the measures of
Congress." The New Jersey Provincial Legislature, in May following,
authorized other townships to appoint delegates for Shrewsbury, but
the same month the refractory township, as will be seen by the
following, chose delegates and also a committee of Observation, and
so the unpleasantness ended. Shrewsbury Falls Into Line. " At a
meeting of Freeholders and Inhabitants of the the township of
Shrewsbury this 27tli day of May 1775, the following persons were
b}^ a great majority, chosen a committee of observation for the said
town agreeable to
 56 HISTORY OF MONMOUTH AND OCEAN COUNTIES. the
direction of the General Continental Congress held at Philadelphia
September 5th, 1774 viz. Josiah Holmes John Little Jos.
Throckmorton Samuel Longstreet Nicholas Van Brunt David Knott
Cor. Vanderveer Benjamin Dennis Daniel Hendrickson Samuel Breese
Thomas Morford Garret Longstreet Cornelius Lane. " Ordered : That
Daniel Hendrickson and Nicholas Van Brunt, or either of them, do
attend the Provincial Congress now setting at Trenton, with full
power to represent there, this town of Shiewsbury. And that Josiah
Holmes, David Knott and Samuel Breese be a sub-committee to
prepare instructions for the Deputy or Deputies who are to attend
the Congress at Trenton. " Josiah Holmes was unanimously chosen
chairman. Josiah Holmes. " Chairman and Town Clerk." Freehold
Patriots Indignant. — Novel Proceedings. March 6th, 1775. A Tory
pamj3hlet entitled " Free Thoughts an the Resolves of Congress ly A.
W. Farmer,''' was handed to the Freehold Committee of Observation
and Inspection for their opinion. The committee declared it to be
most pernicious and malignant in its tendencies and calculated to
sap the foundation of American liberty. The pamphlet was handed
back to their constituents who gave it a coat of tar and turkey
buzzard's feathers, one person remarking that " although the
feathers were plucked from the most stinking of fowls, he thought it
fell far short of being a proper emblem of the author's odiousness to
the friends of freedom and he wished he had the pleasure of giving
the author a coat of the same material." The pamphlet in its
gorgeous attire was then nailed to the pillory post. The same
committee severely denounced a Tory pamphlet written by James
Eivington, editor of Riving 
 BOSTON ACKNOWLEDGES MONMOUTH CONTRIBUTIONS.
57 ton's Royal Gazette, the Tory paper, printed in NewYork. By the
following resolves it will be seen that the citizens of Upper Freehold
favored arming the people if necessary, to oppose the tyrannical acts
of Great Britain. A striking illustration of the stirring events of that
perilous time is found in the fact that before a year had elapsed
some of the prominent men in this meeting were aiding Great Britain
to the best of their ability by voice, pen, or sword : Upper Freehold
Eesolutions. " May 4th 1775. This da^^ agreeable to previous notice
a very considerable number of the principal inhabitants of this
township met at Imlaystown. " John Lawrence Esq. in the chair :
When the following resolves were unanimously agreed to : "
Resolved, That it is our first wash to live in unison with Great Britain,
agreeable to the principles of the Constitution ; that we consider the
unnatural civil war which we are about to be forced into, with
anxiety and distress but that we are determined to oppose the novel
claim of the Parliament of Great Britain to raise a revenue in America
and risk every possible consequence rather than to submit to it. "
Resolved. That it appears to this meeting that there are a sufficient
number of arms for the people. " Resolved. That a sum of money be
now raised to piirchase what further quantity of Powder and Ball
may be necessary ; and it is reccom mended that every man capable
of bearing arms enter into Companies to train, and be prepared to
march at a minute's warning ; and it is further recommended to the
people that they do not waste their powder in fowling and hunting. "
A subscription was opened and one hundred and sixty pounds
instantly paid into the hands of a person appointed for that purpose.
The officers of four companies were then chosen and the meeting
broke up in perfect unanimity. "Elisha Lawrence, Clerk."
 58 HISTOllY OF MONMOUTH AND OCEAN COUNTIES.
INDIAN CLAIMS IN MONMOUTH, OCEAN AND YICINITY. Tlie last
lands in Old Monmoiitli claimed by tlie Indians ware described in
certain papers, p.owers of attorney, &c., presented to a conference
between the whites and Indians held at Cross wicks, N. J., in
February, 1758, For several years previous the Indians had
expressed much dissatisfaction because they had not received pay
for several tracts of land, some of them of considerable extent in
Monmouth and other counties. When the ill feeling of the Indians
became apparent, the Legislature appointed commissioners to
examine into the causes of dissatisfaction. Several conferences were
held at Crosswicks, Burlington, Eastou, Pa., &c., between the
commissioners and the representatives of several Indian tribes with
reference to the lands, and satisfactory settlements made. In the
year 1678, a claim was brought by the Indians against Richard
Hartshorne, an early settler of old Monmouth, who had previously
bought of them Sandy Hook, and lands around the Highlands. In
that 3'ear, to prevent their trespassing upon his lauds, he had to pay
them to relinquish their claims to hunt, fish, fowl, and gather beach
plums. The following is a copy of the agreement: " The 8th of
August, 1678. Whereas the Indians pretend that formerly, when they
sold all the land upon Sandy Hook, they did not sell, or did except
liberty to plums, or to say the Indians should have liberty to go on
Sandy Hook, to get plums when the please, and to hunt upon the
land, and fish, aud to take dry trees that suited them for cannows.
Now know all men by these presents, that I, Richard Hartshorne, of
Portland, in the county of Monmouth, in East Jersey, for peace and
quietness sake, and to the end there may be no cause of trouble
with the Indians and that I may not for the future have any trouble
with them as formerly I had, in their dogs killing my sheep, and their
hunting on my lands, and their fishing, I have agreed as followeth ;
 INDIAN CLAIMS IN MONMOUTH, OCEAN AND VICINITY.
5*J " These presents witnessetli, that I, Vowavapon, Hendricks, the
Indians sonn, having all the liberty and privileges of pluming on
Sandy Hook, hunting, fishing, fowling, getting cannows &c., by these
presents, give grant, bargain, sell, unto Richard Hartshorne, his heirs
and assigns forever, all the liberty and privilege of plum■ ing, fishing,
fowling, and hunting, and howsoever reserved and excepted by the
Indians for him, the said Richard Hartshorne, his heirs and assigns,
to have, hold, possess, and enjoy forever, to say that no Indian, or
Indians, shall or hath no pretense to lands or timber, or liberty,
privileges on no pretense whatsoever on any jjart a parcell of land,
belonging to the said Richard Hartshorne, to say Sandy Hook or land
adjoining to it, in consideration the said Hartshorne, hath paid unto
the said Vowavapon, thirteen shillings money ; and I the ' said
Vowavapon, do acknowledge to have received thirteen shillings by
these presents. Witness my hand and seal " Vowavapon X his mark "
Tocus X his mark. " Signed, sealed and delivered in the presence of
John Stout." Having delivered their claims to the Commissioners, the
Indians present executed a power of attorney to Tom Store, Moses
Totamy, Stephen Calvin, Isaac Still and .John Pompshire, or the
major part of them, to transact all future business with the state
government respecting lands. In 1757 the government had
appropriated £1,600 to purchase a release of Indian claims; one-half
to be laid out in purchasing a settlement for the Indians on the
south side of the Raritau, whereon they might reside ; the other half
to purchase latent claims of back Indians not resident in the
province. At the conference at Easton, in October, 1758, it was
decided to purchase a tract of land in Evesham township, Burlington,
containing over 3,000 acres, for the Indians to locate upon. There
was there a sawmill and cedar swamp and satisfactory hunting
 60 HISTORY OF MONMOUTH AND OCEAN COUNTIES.
ground. The Indians soon removed to this reservation, named
Brotherton ; in removing their buiklijigs thev were assisted by
government. A house of worship and several dwellings were soon
put up. In 1765, it is said, there were about sixty persons settled
there. About the last remnant of Indians remaining in our state, sold
their lands to the whites about 1801, and the year following
removed to New Stockbridge, near Oaeida Lake, New York, from
whence, about 1824, they removed to Michigan, where they
purchased a tract of land of the Menomonie Indians, on both sides
of the Fox river near Green Bay. In 1832, the New Jersey tribe,
reduced to less than forty souls, delegated one of their number
named Bartholomew S. Calvin, to visit Trenton and apply to our
Legislature for remuneration for hunting and fishingprivileges on
unenclosed lands, which they alleged had not been sold with the
land. Calvin was an aged man who had been educated at Princeton,
where he was at the breaking out of the Revolution when he joined
the American army. The claim, so unusual, was met in a spirit of
kindness by our Legislature, who directed the State Treasurer to pay
to the agent of the Indians, the sum of two thousand dollars, thus
satisfactorily and honorably extinguishing the last claim the Indians
brought against our state. Hon. Samuel L. Southard, at the close of
a speech made at the time, said: "It was a proud fact in the history
of New Jersey, that every foot of her soil had been obtained from
the Indians by fair and voluntary purchase and transfer, a fact that
no other state of the Union, not even the land which bears the name
of Penn, can boast." MEMBERS OF THE NEW JERSEY PROVINCIAL
ASSEMBLY FROM MONMOUTH COUNTY. FROM THEIR FIRST
SESSION BEGAN NOVEMBER IOtH, 1703, AT PERTH AMBOY, TO THE
REVOLUTION. In the list of members of the Assembly, or "House
 MEMBEES OF THE NEW JERSEY PROVINCIAL ASSEMBLY. 61
of Representatives of the Province of Nova Cesarea or New Jersey,"
from 1703 to 1709, during which time there were four sessions, the
names of the counties to which they severally belonged are not
given. The records simply mention that they are from East or West
Jersey as the case may be. Among the members from East Jersey it
is probable that the following are from Monmouth County: 1st
Assembly, 1703, Obadiah Bowne, Richard Hartshorne. nn It 1704. *
Richard Hartshorne, John Bowne, I Richard Salter, Obadiah Bowne.
rj n , ( . „^„ \ John Bowne, "William Lawrence, ' ( Lewis Morris. 4th "
1708-9, Gershom Mott, Elisha Lawrence. After this session the
names of the counties to which the members belonged are given.
5th Assembly, 1709, Elisha Lawrence, Gersham Mott. 6th " 1710,
Gershom Mott, William Lawrence, 7th, " 1716, William Lawrence,
Elisha Lawrence. 8th, " 1721, William Lawrence, Garret Schenck.
9th, " 1727, John Eaton, James Grover. 10th, " 1730, John Eaton,
James Grover. llth, " 1738, John Eaton, Cornelius Vandervere. 12th, "
1740, John Eaton, Cornelius Vandervere. 13th, " 1743, John Eaton,
Robert Lawrence. 14th, " 1744, John Eaton, Robert Lawrence. 15th,
" 1745, John Eaton, Robert Lawrence. 16th, " 1746, John Eaton,
Robert Lawrence. 17th, " 1749, John Eaton, Robert Lawrence. 18th,
" 1751, Robert Lawrence, James Holmes. 19th, " 1754, Robert
Lawrence, James Holmes. 20th, " 1761, James Holmes,* Richard
Lawrence. 21st, " 1769, Robert Hartshorne, Edward Taylor. 23d "
1772, Edward Taylor, Richard Lawrence. Robert Lawrence was
speaker of the Assembly in 1746-7, and again from 1754-1758. THE
PROVINCIAL CONGRESS OF NEW JERSEY. The delegates appointed
by the several counties to take action in regard to the tyrannical acts
of Great Britain, assembled at New Brunswick, July 21st, 1774,
*James Holmes died and John Anderson was chosen in his place.
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