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RESEARCH ARTICLE

NF±kB kinetics predetermine TNF-a sensitivity of

colorectal cancer cells

Ralf M. Zwacka*

Lesley Stark

Malcolm G. Dunlop

Department of Oncology, University

of Edinburgh, Edinburgh, UK

*Correspondence to: Ralf M. Zwacka,

Department of Oncology, University

of Edinburgh, Western General

Hospital, MRC Human Genetics Unit,

Crewe Road, Edinburgh EH4 2XU,

UK.

E-mail: [email protected]

Received: 23 May 2000

Revised: 19 June 2000

Accepted: 28 June 2000

Published online: 4 July 2000

Abstract

Background Tumour necrosis factor (TNF)-a has considerable anti-tumour

activity and may have potential as a treatment for metastatic colorectal

cancer. However, TNF-a responses in patients and cell lines are variable and

TNF-a treatment is associated with dose limiting clinical toxicity. Activation of

NF±kB is protective against TNF-a induced cell death, and this may explain

tumour resistance.

Methods In order to provide further understanding of determinants of TNF-

a responses, we studied TNF-a induced NF±kB activation and variable tumour

responses. We analysed the kinetics of TNF-a induced NF±kB activation in

colorectal cancer cells and determined whether it is possible to sensitize

colorectal tumour cells to TNF-a by modulation of NF±kB signalling.

Results We demonstrated that sustained NF±kB activation exceeding 16 h

was observed in HRT18 and SW480 cells and was associated with TNF-a

resistance. In contrast, transient NF±kB activation in HCT116 cells was

associated with sensitivity to cytotoxic TNF-a effects, suggesting that NF±kB

kinetics may have utility as clinical marker of TNF-a tumour resistance.

Despite variable TNF-a responses and NF±kB kinetics, all three colorectal

cancer cell lines were highly sensitive to treatment with the TNF-related

apoptosis-inducing ligand (TRAIL) which induced only transient NF±kB

activation. This further supports the notion of a pre-determined NF±kB

response in¯uencing receptor-mediated cell death. We also show that stable

transfection and adenoviral-mediated expression of IkB(A32/36) can be used

to confer TNF-a sensitivity to colorectal tumour cells previously resistant.

Conclusions These ®ndings indicate that a combined approach using gene

therapy and recombinant TNF-a merits further appraisal. Furthermore, the

kinetics of the TNF-a response could be determined using a `test-dose' to

indicate whether individual patients might bene®t from this gene therapy

approach. Copyright # 2000 John Wiley & Sons, Ltd.

Keywords NF±kB; TNF-a; colorectal cancer; TRAIL; gene therapy

Introduction

Current treatment regimens for hepatic colorectal metastases rarely effect a

cure and only marginally prolong survival. Hence, there is a clear need for

novel approaches to treat such established disease. Combination chemo-

therapeutic approaches that incorporate gene therapy are attractive since

genetic manipulation has the potential to sensitize cancer cells. Initial

excitement surrounding the use of tumour necrosis factor-a (TNF-a)asan

THE JOURNAL OF GENE MEDICINE

J Gene Med 2000; 2: 334±343.

anti-neoplastic agent [1,2] waned as it became clear that

some tumours are relatively resistant to TNF-a and its use

is limited by toxicity [3±5].

Recently, a molecular mechanism, which can account

for at least part of the observed TNF-a resistance has

become evident [6±8]. TNF-a induces a pro-apoptotic

molecular cascade mediated by death domain proteins

that associate with the TNF-a receptor [9]. This results in

activation of a cascade of caspase proteases [10].

However, tumour cells also initiate a protective anti-

apoptotic pathway mediated by activation of NF±kB.

Thus, the ability of TNF-a to elicit cell death depends on

the balance between apoptotic and protective signals.

Several NF±kB target genes such as A20, MnSOD, the bcl-

2 homologue b¯-1/A1 as well as TRAF-1 and 2, in

conjunction with c-IAP-1 and 2, have been implicated in

the anti-apoptotic functions. Overexpression of these

genes has been shown to protect cells from TNF-a

cytotoxicity [11±13]. Since NF±kB activation plays such

a pivotal role in cellular protective responses it might also

have a role in determining TNF-a resistance in colorectal

and other cancer cells. Furthermore, NF±kB responses

may have relevance to drug resistance in other chemo-

therapeutic approaches such as CPT11 [14].

TNF-related apoptosis-inducing ligand (TRAIL) is

another factor shown to activate NF±kB and to induce

cell death in several tumour cell lines [15], but seems to

induce very limited NF±kB mediated anti-apoptotic

responses [16]. It has also been shown to have anti-

tumour activity in xenografts of human colon cancer cell

lines [17]. TRAIL (also known as Apo2), induces

programmed cell death following binding to surface

receptors [18] by utilizing the same cellular death

pathway as TNF-a [19].

The NF±kB transcription factor [20] is normally

sequestered in the cytoplasm by IkB±a, or other members

of the IkB family [21]. Following stimulation by

extracellular signals such as TNF-a, IL-1 or LPS, IkBis

phosphorylated at serine residues 32 and 36 by the IKK-

(IkB±a kinase) complex [22±24]. Such phosphorylation

leads to proteolytic degradation of IkB which in turn

unmasks a nuclear target sequence within NF±kB and

leads to its translocation to the nucleus. The expression of

a super-repressor form of IkB±a mutated at residues 32

and 36, designated IkB(A32/36) or IkB-SR has been

shown to block the nuclear translocation of NF±kBin

Hela, Jurkat and ®brosarcoma cells leading to increased

TNF-a sensitivity [6±8,14]. Inhibition of NF±kB by this

super-repressor prevents the activation of anti-apoptotic

genes such as cIAP-1 and 2 that block a pro-apoptotic

response by inhibition of caspase-8 [13]. Hence, a gene

therapy strategy could be envisaged that exploits mutant

IkB to selectively increase in vivo tumour sensitivity but

with a concomitant TNF-a dose reduction to minimize

clinical toxicity. This strategy requires only transient

expression of IkB (A32/36), and so replication-defective

adenoviral vectors are an attractive gene delivery system.

Several suicide gene approaches are currently in experi-

mental and clinical trials with some degree of success

[25]. Adenoviral mediated IkB (A32/36) tumour trans-

duction followed by TNF-a treatment might have

particular advantages, because TNF-a increases tumour

sensitivity to the chemotherapeutic agent 5-FU [26]. A

combination approach is attractive in view of potential

synergism between agents.

In this article we show that the kinetics of NF±kB

activation have a major in¯uence on the TNF-a response.

Sustained NF±kB activation was associated with TNF-a

resistance and transient NF±kB activation with TNF-a

sensitivity. We provide further evidence that receptor-

mediated apoptosis is in¯uenced by NF±kB activation

kinetics since treatment with TRAIL induced a transient

NF±kB response that resulted in apoptosis in all colorectal

cancer cells studied. The NF±kB response could be used as

a biological marker to predict individual TNF-a responses

with respect to its anti-neoplastic function and could aid

in the tailoring of TNF-a based therapies. We show that

colorectal cancer cells that are resistant to TNF-a can be

sensitized by expression of a dominant negative IkB,

which inhibits the NF±kB mediated anti-apoptotic cellular

programme.

Materials and methods

Cell culture and viral infection

HCT116, HRT18 and SW480 human colorectal cancer

cells were grown in McCoy, RPMI and L-15 medium

(Gibco BRL, Paisley, UK), respectively, containing 10%

fetal calf serum (FCS) and 1% penicillin/streptomycin.

For functional studies, cells were treated with TNF-a

(R&D Systems, Minneapolis, MN, USA) of concentrations

of 0, 1, 10 and 100 ng/ml in 2% serum-containing

medium and harvested at different time-points. TRAIL

(R&D Systems) was used at a concentration of 10 ng/ml.

Stable transfectants were generated by transfection of a

pcDNA construct containing an IkB(A32/36) cDNA under

the control of a CMV early promoter/enhancer element

[27]. A standard lipofection method was used (Gibco).

The transfected cells were selected under 1.5 mg/ml

G418 (Gibco) and 20 individual clones picked, expanded

and tested for transgene expression. Despite several

attempts we have not been able to identify any IkB-

expressing SW480 clones. We believe that such clones

might have signi®cant survival disadvantages during

G418 selection.

Adenovirus infections were performed in medium

containing 2% FCS and 1% penicillin/streptomycin for

24 h, after which cells were harvested for transgene

analysis, or TNF-a treatment was started. Multiplicity of

infection (MOI) of 0, 1000, 5000 and 10 000 particles/

cell corresponding to 0, 40, 200 and 400 pfu/cell (25

particles required to infect one cell of the reference cell

line 293), respectively were used. For growth studies and

EMSA analysis an MOI of 5000 was used. Growth was

measured by seeding 10

cells into each well of a 24-well

plate, and treated with TNF-a, as described above, 2 days

NF-kB Activation Kinetics in TNF-a Resistance 335

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