Inactivation of isocitrate dehydrogenase kinase/phosphatase by 5''-[p-(fluorosulfonyl)benzoyl]adenosine is not due to the labeling of the invariant lysine residue found in the protein kinase family

Previous studies from our laboratory, using p53 transgenic mice, have suggested that ultraviolet (UV) light-induced keratinocyte apoptosis in the skin is not affected by overexpression of mutant p53 protein. To further elucidate a possible role for p53 in UV-induced keratinocyte cell death, we now examine apoptosis in skin and isolated keratinocytes from p53 null (-/-) mice and assess the influence of cell differentiation on this process. In vivo, using this knockout model, epidermal keratinocytes in p53-/- mice exhibited only a 5.2-fold increase in apoptosis after 2000 J/m2 UVB irradiation compared with a 26.3-fold increase in normal control animals. If this p53-dependent apoptosis is important in elimination of precancerous, UV-damaged keratinocytes, then it should be active in the undifferentiated cells of the epidermal basal layer. To test this hypothesis, we examined the effect of differentiation on UV-induced apoptosis in primary cultures of murine and human keratinocytes. Apoptosis was p53-independent in undifferentiated murine keratinocytes, which exhibited relative resistance to UVB-induced killing with only a 1.5-fold increase in apoptosis in p53+/+ cells and a 1.4-fold increase in p53-/- cells. Differentiated keratinocytes, in contrast, showed a 9.4-fold UVB induction of apoptosis in p53+/+ cells, almost three times the induction observed in p53-/- cells. This UV-induced difference in apoptosis was observed when keratinocytes were cultured on type IV collagen substrate, but not on plastic alone. Western blotting of UV-irradiated, differentiated keratinocytes did not support a role for either Bax or Bcl-2 in this process. In support of these findings in mice, cell death in human cultured keratinocytes also occurred in a differentiation-associated fashion. We conclude that p53-induced apoptosis eliminates damaged keratinocytes in the differentiated cell compartment, but this mechanism is not active in the basal, undifferentiated cells and is therefore of questionable significance in protection against skin cancer induction.     

Biologic effects of introduction of wild-type p53 cDNA into a human ovarian cancer cell line SKOV-3

OBJECTIVE: To study the effects on biologic behavior in cells obtained from human ovarian cancer cell line SKOV-3 into which the wild-type p53 cDNA was introduced. METHOD: Recombinant eukaryotic expression vector pC53-SN3 containing full-length human wild-type p53 cDNA and vector containing neomycin resistance gene only were introduced by lipofectamine-mediated gene transfection into SKOV-3 cell line which does not express endogenous p53. The clones obtained were observed for their biologic behavior. RESULTS: (1) 2 clones named pC53 and 2 clones named pNeo were obtained after pC53-SN3 and vector transfection respectively; (2) The morphology of cells either from pC53 or from pNeo did not change significantly with respect to their parental SKOV-3; (3) The growth rate of cells from pC53 was much slower than that from SKOV-3, while the cell growth curve of pNeo was similar to that of SKOV-3; (4) The number of colones formed in the soft-agar by pC53 was significantly less than that by SKOV-3 or by pNeo; (5) The percentage of phase G1/G0 of pC53 was much higher than that of SKOV-3 and pNeo. CONCLUSION: Wild-type p53 cDNA may be considered as one of the target genes for the gene therapy of ovarian cancer.     

EGF receptor signaling inhibits keratinocyte apoptosis: evidence for mediation by Bcl-XL

Signaling through the epidermal growth factor receptor (EGFR) has been primarily implicated in the growth of epithelial cells including keratinocytes. However, the mechanism by which EGFR stimulation promotes keratinocyte cell growth is poorly understood. Here we report that human keratinocytes undergo apoptosis when incubated with the blocking EGFR monoclonal antibody 225 IgG, or PD153035, a highly specific EGFR tyrosine kinase inhibitor. Endogenous mRNA and protein levels of Bcl-XL, a member the Bcl-2 family which suppresses apoptosis, were specifically inhibited by EGFR blockade. Furthermore, stimulation of EGFR signaling through two natural ligands, transforming growth factor (TGF)-alpha and epidermal growth factor (EGF), increased the expression of Bcl-XL in quiescent keratinocytes and HaCaT cells. Finally, ectopic expression of Bcl-XL in HaCaT cells increased survival after EGFR blockade when compared to untransfected cells or HaCaT keratinocytes transfected with empty vector. These results suggest that the anti-apoptotic protein Bcl-XL plays an important role in the maintenance of keratinocyte survival in response to EGFR signaling.     

Structural correlates of cognitive deficits in a selected group of patients with Alzheimer''s disease

Mutated RAS oncoproteins and epidermal growth factor (EGF) are thought to contribute to the proliferative, invasive and metastatic properties of transformed cells. In the present study, we investigated the role of EGF in two H-ras transfected clones and compared it to that in the parental cell line, HaCaT and primary cultured keratinocytes. Our findings show that the motility on type I collagen, measured by the migration index, was similar for both the HaCaT cell line and normal human keratinocytes, whereas it was higher for the HaCaT-ras clones. These results suggest an involvement of the ras oncogene in the stimulation of cell migration. EGF in cell pretreatment or during the migration assay also caused an increase in migration of all the cells, but preserved the difference between HaCaT and HaCaT-ras. However, no significant difference in EGF-R expression was detected between normal cultured keratinocytes, HaCaT and HaCaT-ras cell lines with or without EGF pretreatment. Moreover, when the cells were stimulated with EGF, the MMP-9 activity was greatly increased in a dose-dependent manner in all the cells, and EGF stimulation particularly highlights the increased amount of MMP-9 in HaCaT-ras cells compared to HaCaT cells. In conclusion, EGF is able to enhance motility and to up-regulate MMP-9 activity in all cells, but with a higher impact in HaCaT-ras cells without an overexpression of EGF-R. As EGF acts in synergy with the H-ras mutation, they could be implicated in the local invasion by the HaCaT-ras clones.     

Bronchopulmonary dysplasia and oxidative stress: are we closer to an understanding of the pathogenesis of BPD?

OBJECTIVES: To determine if induction of heat shock protein 70 (HSP 70), a stress protein that plays a cytoprotective role and inhibits cell death in response to various stimuli, will protect thymocytes and T-cell clones from radiation-induced apoptosis, and to define the mechanism of such protection. DESIGN: Thymocytes from BALB/c mice or T-lymphocyte clones were incubated at 43 degrees C for 1 hour to induce HSP 70, then irradiated. Control cells were irradiated but not heated. Fragmentation of DNA was quantitated, and p53, bax, and bcl-2 expression was analyzed at various times by the Western blot method. RESULTS: Only heated cells expressed HSP 70. The induction of HSP 70 increased basal apoptosis but significantly decreased radiation-induced apoptosis. Furthermore, introduction of an HSP 70 antisense oligomer prior to heating reversed the protective effect of HSP 70. Induction of HSP 70 in T-cell clones with sodium arsenite had a similar protective effect against radiation-induced apoptosis. Irradiation induced p53 and markedly up-regulated bax. The expression of p53 peaked at 4 hours and preceded maximal bax induction. Induction of HSP 70 prior to irradiation suppressed p53 and significantly decreased bax levels. Levels of bcl-2 were unaffected. CONCLUSIONS: Our data show that HSP 70 induction protects thymocytes from radiation-induced apoptosis by down-regulating p53 and bax expression. The induction of HSP 70 may represent a novel mechanism by which the immunosuppressive effects and the associated infectious complications of radiation therapy can be minimized.     

WAF1/p21 regulates proliferation, but does not mediate p53-dependent apoptosis in urothelial carcinoma cell lines

The WAF1/p21 gene product is an inhibitor of cyclin-dependent kinases which can be induced by the tumor suppressor p53 and mediate some of its effects, or function in p53-independent pathways of cell cycle regulation. Although a potential tumor suppressor gene, WAF1/p21 is expressed in bladder cancer. To elucidate the function of p21 in tumor cells we have investigated in urothelial carcinoma cell lines: i) WAF1/p21 mRNA and protein expression, ii) the biological effects of p21 overexpression or down-regulation and (iii) whether p21 can be induced by p53. WAF1/p21 mRNA levels examined in four cell lines were comparable to bladder mucosa. One cell line, HT1376, failed to express p21 protein due to a frame shift mutation. Overexpression of WAF1/p21 cDNA inhibited clone formation in three cell lines, whereas transfection with antisense WAF1 increased clone sizes and numbers. WAF1 sense clones showed diminished cell proliferation compared to the parental cell line. Apoptosis- induced wild-type p53 was not inhibited by overexpression of antisense WAF1/p21. In a cell clone derived from line VMCub1 by stable transfection with wild-type p53 under the control of a metallothionein promotor, p21 was induced along with p53 upon activation of the promoter with zinc chloride. This induction was accompanied by a decrease in cell proliferation but by little apoptosis. These data suggest that p21 inhibits proliferation in a p53-dependent or independent manner but does not mediate p53-induced apoptosis in urothelial carcinoma cells.     

Low dose desensitisation does not reduce the toxicity of sulphasalazine in rheumatoid arthritis

To investigate the apoptosis-inducing effect of glucocorticoid on gastric epithelial cell expressing different p53 genes, a human gastric epithelial cell line-GES-1 was transfected with either wild type or mutant p53 cDNA in vitro. The cells were treated with hydrocortisone in a concentration range of 0.2-0.8 g/L. Apoptotic cells were found in those transfectants. The apoptotic response of the wild-type p53-transfected GES-1 cells was much more marked than that of the mutant p53 transfected ones. It can be inferred that the glucocorticoid secreted not only suppress the immunological response during inflammation, but induce gastric epithelial cell apoptosis as well. If the gastric epithelial cell contains mutant p53 genes, the glucocorticoid confer a selective growth advantage which advance the malignant process.     

Transfection of inducible nitric oxide synthase gene causes apoptosis in vascular smooth muscle cells

BACKGROUND: Excess production of nitric oxide (NO) by inducible NO synthase (iNOS) has been implicated in a variety of physiological processes including vascular remodeling. To elucidate whether endogenous NO generated by iNOS is involved in the programmed cell death (apoptosis) of the vasculature, iNOS cDNA- expressing construct was transfected into rat and human vascular smooth muscle cells (VSMCs) by lipofection. METHODS AND RESULTS: VSMCs transiently transfected with iNOS cDNA functionally expressed 130 kd iNOS protein with full catalytic activity to generate massive NO in proportion to the doses of cDNA used; its enzymatic activity as well as NO production was completely blocked by an NOS inhibitor, NG-monomethyl-L-arginine (LNMMA). Overexpression of iNOS led to a marked inhibition of DNA synthesis as well as induction of apoptosis in VSMCs. Evidence for apoptotic cell death was provided by internucleosomal DNA fragmentation by agarose gel electrophoresis, positive staining for TdT-mediated dUTP biotin nick end-labeling, and appearance of hypodiploid cells by flow cytometry analysis. Apoptosis after transfection with iNOS cDNA was abrogated by LNMMA. Transfection of iNOS cDNA caused accumulation of the tumor suppressor gene p53 but not of bcl-2, which was also blocked by LNMMA. CONCLUSIONS: These results demonstrate that massive generation of endogenous NO derived from iNOS overexpression leads to a marked apoptosis in VSMCs, thus suggesting an important role of NO as a proapoptotic factor for VSMCs in the process of vascular remodeling.     

p53 mutation and tamoxifen resistance in breast cancer

A substantial portion of patients with estrogen receptor-positive breast cancer fail to respond to estrogen depletion or to the antiestrogen tamoxifen. The molecular changes that lead to tamoxifen resistance and estrogen-independent growth are unknown. To test the hypothesis that a p53 mutation could result in tamoxifen resistance and estrogen-independent growth, the MCF-7 cell line was transfected with p53 cDNA which was mutated at codon 179 (histidine to glutamine). MCF-7 is an estrogen receptor-positive, estrogen-dependent, tamoxifen-sensitive cell line with only wild-type p53. The presence of transfected mutant p53 cDNA was verified by the PCR, and overexpression of p53 protein was assessed by Western blotting. Five separate mutant-transfected clones were selected and tested in subsequent growth experiments. In monolayer culture, there was no consistent evidence of estrogen-independent growth or tamoxifen resistance in the mutant transfectants compared with vector-only controls or the parental cell line. In soft agar growth experiments, four of five mutant transfectants remained sensitive to tamoxifen in a dose-dependent manner. In the presence of wild-type p53, mutant 179 p53 protein does not result in estrogen-independent growth or tamoxifen resistance. These results do not exclude the possibility that other p53 mutational types could result in tamoxifen resistance, or that loss of the remaining wild-type allele may be necessary to result in this phenotype.     

Cytotoxic effects of adenovirus-mediated wild-type p53 protein expression in normal and tumor mammary epithelial cells

To evaluate the effects of the wild-type p53 expression in normal and tumor cells, we have constructed a recombinant adenovirus vector (E1 minus) expressing human wild-type p53 cDNA (AdWTp53). Infection of normal and tumor cells of lung and mammary epithelial origin with AdWTp53 resulted in high levels of wild-type p53 expression. Production of p53 protein following infection was dependent on the dose of AdWTp53 with maximum amounts of p53 produced following infection with 50 plaque-forming units/cell. AdWTp53 infection inhibited the growth of all human cell lines studied. However, tumor cells that were null for p53 prior to infection (H-358 and MDA-MB-157) and tumor cells that expressed mutant endogenous p53 protein (MDA-MB-231 and MDA-MB-453) were more sensitive to AdWTp53 cytotoxicity than cells that contained the wild-type p53 (MCF-7, MCF-10, 184B5, and normal mammary epithelial cells). All cells exhibited WAF1/Cip1 mRNA and protein induction following AdWTp53 infection. AdWTp53-induced cytotoxicity of human tumor cell lines expressing mutant p53 was mediated by apoptosis as revealed by nucleosomal DNA fragmentation analysis. No detectable nucleosomal DNA fragmentation was observed following AdWTp53 infection of human cells expressing wild-type p53. These data suggest that endogenous p53 status is a determinant of AdWTp53-mediated cell killing of human tumor cells.     

Expression of p53, Bcl-2 and Bax in cisplatin-induced apoptosis in testicular germ cell tumour cell lines

We examined the sensitivity for cisplatin-induced apoptosis in a panel of four testicular germ cell tumour (TGCT) cell lines and monitored the cellular expression of the apoptosis-related proteins p53, Bcl-2 and Bax. Three of four TGCT cell lines (NT2, NCCIT and S2) were hypersensitive for cisplatin-induced apoptosis, while the TGCT cell line 2102 EP appeared to be resistant for cisplatin-induced apoptosis, even at relatively high drug concentrations (12.5 microM). For all four cell lines, the induction of apoptosis by cisplatin correlated with drug sensitivity in the MTT assay. The differences in chemosensitivity and induction of apoptosis could not be attributed to differences in cellular platinum accumulation, DNA platination or platinum-DNA adduct removal. We next analysed the relationship between p53 status and cisplatin-induced up-regulation of p53, and the susceptibility to cisplatin-induced apoptosis. Wild-type p53 containing NT2 and 2102 EP cells showed p53 up-regulation upon drug treatment, and NCCIT (mutant p53) and S2 (no p53 protein) cells did not. Consistently, the increase in wild-type p53 protein in NT2 and 2102 EP cells led to an increase in mRNA level of the p53 downstream gene p21/WAF/CIP, whereas mutant p53-containing NCCIT cells and p53-non-expressing S2 cells could not transactivate this p53-responsive gene. As NT2, NCCIT and S2 were readily triggered into apoptosis, while 2102 EP cells failed to undergo cisplatin-induced apoptosis, our data suggest that the presence of wild-type and/or transactivation-competent p53 might not be an absolute prerequisite for efficient induction of apoptosis in TGCT cell lines. Also endogenous levels of Bcl-2 and Bax expression did not correlate with cisplatin-induced apoptosis. In addition, the endogenous Bcl-2 and Bax expression was not affected by cisplatin treatment. The present study suggests that, at least in our panel of TGCT cell lines, hypersensitivity for cisplatin-induced apoptosis might not be necessarily correlated with the presence of wild-type p53 and is probably not associated with Bcl-2 and Bax expression.     

Radioresistant MTp53-expressing rat embryo cell transformants exhibit increased DNA-dsb rejoining during exposure to ionizing radiation

Recent data suggest that aberrant function of the wild type p53 protein (WTp53) may alter cellular survival following DNA damage through cellular pathways involving apoptosis and cell-cycle checkpoints, but little is known concerning it's possible role in DNA repair. In the present study, the ionizing radiation sensitivity was determined for a series of rat embryo fibroblast (REF) cell lines transfected with an activated form of the H-ras oncogene alone, or in combination with a variety of missense-mutant p53 (MTp53) alleles. Transformed REF clones which expressed exogenous MTp53 and p21ras proteins (CLASS II clones) were generally radioresistant in culture as determined by higher values for the surviving fraction after 2 Gy (SF2 value) and the radiation dose required to reduce survival to a fraction of 0.1 (D10 value), compared either to transformed REF clones expressing p21ras protein alone (CLASS I clones), or to non-transfected REF control cell lines expressing baseline endogenous levels of p21ras and WTp53 protein. The increased radioresistance observed in the CLASS II clones (following both HDR- and LDR-irradiation), was significantly correlated with increased expression of MTp53 protein, and a decreased radiation-induced G1 arrest response. The variability observed in clonogenic radiosensitivity among REF clones was not explained by differential radiation-induced apoptosis. Using the Comet assay performed after continuous low dose-rate (LDR)-irradiation, MTp53-expressing REF clones were also found to be more proficient at the rejoining of DNA double-strand breaks (DNA-dsb), compared to WTp53-expressing REF clones. These results suggest that an enhanced DNA and cellular repair capacity may, in part, explain the increased radiation survival observed in some MTp53-expressing transformed fibroblasts and tumours.     

Growth arrest and suppression of tumorigenicity of bladder-carcinoma cell lines induced by the P16/CDKN2 (p16INK4A, MTS1) gene and other loci on human chromosome 9

Wild-type P16/CDKN2 (p16INK4A, MTS1) cDNA, directed by the cytomegalovirus (CMV) immediate early promoter, was transfected into RT4 and RT112 bladder-carcinoma cell lines bearing a mutated endogenous P16/CDKN2 gene and lacking endogenous P16/CDKN2 respectively. In both cases, only transfected clones with rearranged exogenous P16/CDKN2 cDNA could be grown and propagated in cell culture. This result is reminiscent of transfection of wild-type p53 into cells with a deleted or mutated endogenous gene and suggests that P16/CDKN2, over-expressed under control of the strong CMV promoter, induces growth arrest in RT4 and RT112 cells. Transfer of human chromosome 9 to RT4 cells produced RT4/H9 hybrid clones retaining the P16/CDKN2 gene, since in RT4/H9 cell clones P16/CDKN2-gene expression is modulated by the physiological control of chromosomal regulatory sequence. All the RT4/H9 clones lost the entire chromosome 9, except clone 4 and clone 5, which maintained a deleted and an intact chromosome 9 respectively. Loss of several loci in 9p21, including P16/CDKN2, in tumors induced in nude mice by clone 4 and clone 5 suggests that P16/CDKN2 or other genes in 9p21 suppress tumorigenicity in bladder-carcinoma cells. Tumors induced by clone 4 and clone 5 show loss of markers in 9q. The regions 9q22.3, 9q32-33 and 9q34.2, which were maintained in the 2 clones and lost in their derived tumors, may contain tumor-suppressor genes relevant in bladder carcinoma. The results of this study suggest that the P16/CDKN2 gene controls growth of bladder-carcinoma cells when it is over-expressed, and may be involved in the development of bladder carcinoma, but other genes in 9p21 and 9q may participate in bladder-cancer progression.     

Apoptosis, reproductive failure, and oxidative stress in Chinese hamster ovary cells with compromised genomic integrity

Chromosomal instability and persistent reproductive cell death show a significant correlation after cells are exposed to ionizing radiation. To examine the possible role of apoptosis in persistent reproductive cell death, we analyzed subsets of chromosomally stable and unstable clones for relationships between chromosome stability, reproductive integrity, and apoptosis. All clones were generated from the GM10115 cell line and derived from single progenitor cells surviving 10 Gy of X-rays, and all measurements were made approximately 60-80 generations after irradiation. The incidence of apoptosis, as measured by both annexin V binding of phosphatidylserine residues and terminal deoxynucleotidyl transferase labeling of DNA strand breaks, was significantly higher in chromosomally unstable clones than it was in chromosomally stable clones (P < 0.05; ANOVA and Student's t test). Furthermore, statistical analyses of the biological end points of persistent reproductive cell death and apoptosis were consistent, showing R2 values of 0.78 and 0.76, respectively. These results suggest that persistent reproductive cell death can, in part, be explained by the predisposition of a fraction of the clonal population to undergo apoptosis or necrosis. Immunological blot analyses of protein levels and DNA bandshift assays confirmed the mutant status of p53 in the host cell line, implying an apoptotic pathway that is independent of p53. Induction of apoptosis by agents such as actinomycin D, etoposide, and staurosporine and induction of necrosis by sodium azide were accompanied by an increase in the level of intracellular peroxy radicals and lipid peroxidation products, two independent end points that are typically associated with oxidative stress. Similar findings were observed in several subclones showing persistent apoptosis. These results suggest that the elevated levels of free radical damage that we detected were derived from the fraction of cells dying by apoptotic or necrotic processes. Possible mechanisms whereby oxidative stress may contribute indirectly to the perpetuation of chromosomal instability are discussed.     

Abrogation of c-Raf expression induces apoptosis in tumor cells

Signal transduction pathways involving the c-Raf protein kinase are frequently activated in tumor cells. We have addressed the relevance of this activation by a loss-of-function approach. An anti-sense phosphorothioate oligonucleotide (ODN) specifically targeted against c-raf mRNA (Monia et al., 1996a) was used to block c-Raf protein expression in four different cell lines derived from lung, cervical, prostate and colon carcinomas. Concomitant with the abrogation of c-Raf expression we observed the occurrence of classical apoptotic markers, including chromatin condensation, inter-nucleosomal DNA cleavage, annexin V binding and cleavage of PARP, which was followed by cell death, affecting most of the cell population. This induction of apoptosis occurred independent of the p53 status of the cell. These findings demonstrate that c-Raf can protect tumor cells from undergoing programmed cell death, and suggest that the interference with c-Raf expression or function by ODNs or specific drugs could represent a powerful means for improving the efficacy of anti-cancer therapy.     

Expression of wild-type p53 increases etoposide cytotoxicity in M1 myeloid leukemia cells by facilitated G2 to M transition: implications for gene therapy

We have evaluated the role of p53 in the induction of cell death by the DNA topoisomerase II inhibitor etoposide in M1 myeloid leukemia cells. Three different clones of M1 cells were used: S6, which lacks p53; Phe-132, which expresses mutant p53 constitutively; and LTR-13, which expresses mutant protein at 37 degrees C and wild-type p53 at 32 degrees C. As described previously, LTR-13 cells undergo rapid apoptosis upon induction of wild-type p53 at 32 degrees C. Multiparameter flow cytometric analysis showed that etoposide treatment (0.5 microg/ml) of all three cell lines at 37 degrees C is associated with a block in the G2 phase of the cell cycle, whereas the cells preferentially die out of the next S phase. Induction of wild-type p53 in LTR-13 cells is associated with a loss of cells in late S and G2-M phase, and the cells die out of the early S phase. Interestingly, the simultaneous induction of apoptosis by both pathways (wild-type p53 and etoposide) leads to suppression of the etoposide-induced G2 block. To determine the effect of p53 on the G2 to M transition, LTR-13 cells were incubated with etoposide for 24 h at 37 degrees C and then either maintained for an additional 12 h at 37 degrees C or shifted to 32 degrees C to activate wild-type p53. The expression of wild-type p53 resulted in an increase in mitosis-specific phosphorylation, as determined by the MPM-2 antibody as well as the formation of mitotic spindles. This was associated with an important augmentation of the cytotoxic effect of etoposide. In contrast, a similar temperature shift of Phe-132 cells, which express mutant p53, had no effect on either immunostaining with MPM-2 or the cytotoxicity. Taken together, our results indicate that wild-type p53 can override the etoposide-induced G2 block in at least some cell types. These data propose a new role for p53 in the cell death induced by chemotherapeutic agents and may have important implications for gene therapy.     

Hprt- mutation spectrum in a closely related pair of human bladder tumour cell lines after gamma-irradiation at different dose-rates

Inactivation of p53 gene and overexpression of MDR1 gene are both associated with drug resistance. Previous studies have suggested that p53 gene can modulate the expression activity of MDR1 gene promoter in a promoter-CAT system. In the present study, wild-type p53 gene cDNA was introduced into a multidrug-resistant cell line, KBv200, in which endogenous p53 gene is aberrant. In wt-p53 transfected cells, the expression of MDRI gene was significantly increased, accumulation of adriamycin (ADM) was decreased, and the sensitivity to vincristine (VCR), ADM and 5-fluorouracil (5-FU) was increased compared with the parent KBv200 cells. After treatment with ADM and VCR, the p53-transfectants were more susceptible to apoptosis. The results suggest that the increase in drug sensitivity of the cells may be, at least in part, due to p53-dependent apoptosis induced by anticancer agents.     

Prevention of tumor necrosis factor (TNF)-mediated induction of p21WAF1/CIP1 sensitizes MCF-7 carcinoma cells to TNF-induced apoptosis

The MCF-7 breast carcinoma and MRC-5 lung fibroblast cell lines are sensitive and resistant to tumor necrosis factor (TNF)-induced apoptosis, respectively. As the cyclin-dependent kinase inhibitor p21WAF1/CIP1 (p21) is involved in cell cycle regulation and has been implicated in apoptosis, we studied the influence of p21 on growth of MRC-5 cells and on growth and apoptosis in MCF-7 cells. TNF induced p21 mRNA and protein in both cell types. p21 induction by > 0.5 ng/ml TNF in MRC-5 and MCF-7 cells correlated with the inhibition of cell growth. In contrast, < 0.1 ng/ml TNF stimulated MRC-5 (but not MCF-7) cell growth without reduction in p21 levels. TNF-induced apoptosis in MCF-7 cells was first detected after the TNF-mediated increase in p21 and growth arrest had occurred. MCF-7 cells stably transfected with antisense p21 cDNA became more sensitive to TNF-induced apoptosis. Thus, TNF-induced p21 accompanied by growth arrest may counteract or delay TNF cytotoxicity in MCF-7 cells.