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J C Reed

Publications and source records attributed to J C Reed.

At least 325 records · Page 18Linked to original sources
PubMed1996

Increased intestinal Bak expression results in apoptosis.

Cells in the human intestinal epithelium have a life-span of around 5 days before being lost by apoptosis at the luminal surface. We examined changes in expression of the Bcl-2 gene family which may be responsible for epithelial cell loss. In the normal and neoplastic colon, mucosal expression of immunoreactive Bak co-localized with sites of epithelial cell apoptosis. Inducing apoptosis in the human colon cancer cell line HT29 and the rat normal small intestinal cell line IEC 18 in culture was accompanied by increased Bak expression without consistent changes in expression of other Bcl-2 homologous proteins. Bak appears to be the endogenous Bcl-2 family member best correlated with intestinal cell apoptosis.

Animals↗
PubMed1996

Bcl-2 interrupts the ceramide-mediated pathway of cell death.

Ceramide, a product of sphingomyelin turn-over, has been proposed as a novel lipid second messenger with specific roles in mediating antiproliferative responses including apoptosis and cell cycle arrest. In this study, we examine the relationship between the ceramide-mediated pathway of growth suppression and the bcl-2 protooncogene. In ALL-697 leukemia cells, the addition of the chemotherapeutic agent vincristine resulted in a time-dependent growth suppression characterized by marked apoptosis. The effects of vincristine on cell death were preceded by a prolonged and sustained accumulation of endogenous ceramide levels reaching -10.4 pmol ceramide/nmol phospholipids at 12 hr following the addition of vincristine--an increase of 220% over vehicle-treated cells. Overexpression of bcl-2 resulted in near total protection of cell death in response to vincristine. However, the ceramide response to vincristine was not modulated by overexpression of bcl-2, indicating that bcl-2 does not interfere with ceramide formation. Overexpression of bcl-2 prevented apoptosis in response to ceramide, suggesting that bcl-2 acts at a point downstream of ceramide. On the other hand, bcl-2 did not interfere with the ability of ceramide to activate the retinoblastoma gene product or to induce cell cycle arrest, suggesting that the effects of ceramide on cell cycle arrest can be dissociated from the effects on apoptosis. These studies suggest that ceramide and bcl-2 partake in a common pathway of cell regulation. The results also cast ceramide as a gauge of cell injury rather than an "executor" of cell death with clearly dissociable biological outcomes of its action depending on downstream factors.

Antineoplastic Agents, Phytogenic↗
PubMed1996

Acute and delayed apoptosis induced by thymidine deprivation correlates with expression of p53 and p53-regulated genes in colon carcinoma cells.

The endogenous expression of p53 and p53-regulated genes has been examined in a thymidylate synthase-deficient colon carcinoma cell line (TS-) and a derived mutant clone (Thy4) that exhibit acute or delayed apoptotic responses, respectively, when released from G0 synchrony under conditions of dThd starvation. These cell clones demonstrate heterozygosity in p53, thereby expressing one wt allele and one with an A-->C point mutation at codon 240. Following release from G0, upregulated expression of both alleles occurred. During apoptosis in TS-, a wtp53 phenotype was expressed and in Thy4 during cytostasis, a mp53 phenotype was manifested, as determined from the ratios of wtp53/mp53 proteins, transactivation of p50-2 (a wtp53-responsive CAT reporter construct) and the endogenous expression of MDM2. Neither cytotoxicity nor cytostasis correlated with expression of p21Waf1/Cip1 Thy4 cells sustained accumulation of high levels of Bax in a wtp53-independent and dThd-independent manner and survival was associated with upregulated expression of Bcl-2. In contrast, Bax expression decreased in TS- during apoptosis, except in a highly resistant subpopulation that retained high levels of Bax. Data suggest that resistant cells (Thy4) can sustain high Bax expression and that Bcl-2 is upregulated in response to an apoptotic stimulus due to the absence of negative regulation by wtp53.

Alleles↗
PubMed1996

Elevated expression of Bcl-X and reduced Bak in primary colorectal adenocarcinomas.

Expression of several members of the BCL-2 family of genes was investigated by immunohistochemical methods in 30 primary colorectal adenocarcinomas and 24 adenomatous polyps. When compared to the intensity observed in adjacent normal mucosal epithelial cells, the intensity of Bcl-X immunostaining was elevated in 18 of 30 (60%) carcinomas (P = 0.0001) and 12 of 24 (50%) adenomatous polyps (P = 0.0001). Immunoblot analysis of five pairs of tumors and adjacent normal colonic tissue indicated marked elevations in the relative levels of the anti-apoptotic Bcl-XL, protein in all cases. In contrast to the increased Bcl-X expression, the intensity of Bcl-2 immunostaining was greater than that of normal colonic mucosa in only 3 of 30 (10%) carcinomas and, in fact, was lower than that of adjacent normal epithelia] cells in 25 (83%) cases (P = 0.0001). Furthermore, the percentage of Bcl-2 immunopositive cells was generally lower in carcinomas than in adenomas (mean +/- SE, 44 +/- 6% versus 73 +/- 5%, respectively; P = 0.001) and in moderately or poorly differentiated tumors than in well-differentiated tumors (39 +/- 6% versus 70 +/- 11%, respectively; P = 0.045). In addition, the proportion of tumors in which the Bcl-2 immunointensity was more than or equal to that of normal colonic mucosa was significantly lower in carcinomas than adenomas (5 of 30 versus 15 of 24, respectively; P < 0.001), suggesting that decreases in Bcl-2 expression represent a later event associated with the progression of colorectal cancers. When compared to that of normal adjacent colonic epithelium, the intensity of Mcl-1 immunostaining was reduced in 20 of 30 (67%) of carcinomas (P = 0.0001) compared to only 1 of 24 adenomas, suggesting that decreases in Mcl-1 expression represent a later event associated with progression from a benign to a malignant phenotype or with transition to a less-differentiated state, because most of the carcinomas evaluated here (25 of 30; 83%) were not well differentiated. The intensity of immunostaining for the pro-apoptotic protein Bak was reduced compared to that of normal mucosal epithelial cells in 27 of 30 (90%) carcinomas and 22 of 24 (92%) adenomas, suggesting that reductions in Bak expression occur early in colorectal tumor progression (P = 0.0001). In contrast, the intensity of immunostaining for the pro-apoptotic protein Bax was not significantly altered in carcinomas; compared to that of normal colonic mucosa, Bax immunointensity was reduced in only 7 of 30 (23%) carcinomas and 3 of 24 (13%) adenomas, and the percentage of Bax immunopositive cells was also not significantly different in any of the histological subgroups. Taken together, these results suggest that expression of Bcl-XL is increased in undifferentiated primary colorectal cancers, often with accompanying reciprocal decreases in the anti-apoptotic proteins Bcl-2 and Mcl-1 and the pro-apoptotic protein Bak, whereas Bax expression is relatively constant. Thus, a shift from expression of the anti-apoptotic proteins Bcl-2 and Mcl-1 to the Bcl-XL protein may occur during progression of colorectal tumors.

Adenocarcinoma↗
PubMed1996

Detection of multiple antigens on western blots.

An immunoblotting method is described that permits sequential detection of multiple antigens on a single protein blot without stripping off prior antibodies. The procedure utilizes horseradish peroxidase (HRPase)-based detection with a chemiluminescent substrate. After detection by enhanced chemiluminescence (ECL) with exposure to X-ray film, the antigen-antibody complexes on the blot are reacted with a chromogenic substrate (either 3.3'-diaminobenzidine [DAB] or SG [Vector Labs, Inc.]) which renders the antigen-antibody-HRPase complexes unreactive in subsequent reprobings of the same membrane with additional antibodies using the same detection method. Because no stripping is involved, immobilized proteins are not lost from the membrane, thus allowing for multiple sequential reprobings of the same membrane with different primary antibodies (> or = 12) and retention of strong signal intensities for all antibody probings. A variation of the method (the "rainbow Western") is described in which four different HRPase-colorimetric substrates that produce black, brown, red, and green colors are employed sequentially for detection and simultaneous display of four different antigens on the same blot. Both techniques could be particularly valuable for analysis of cellular populations that are difficult to isolate in large numbers or of clinical specimens where the amounts of protein samples that can be obtained are limiting or only available on a one-time basis.

Antigens↗
PubMed1996

Apoptotic response to oncogenic stimuli: cooperative and antagonistic interactions between c-myb and the growth suppressor p53.

c-myb, a protooncogene prevalently expressed in the hematopoietic tissue, is a transcription factor that contains a DNA-binding domain and an acidic domain and is able to transactivate specific viral and cellular genes. In this report, we show that c-myb can stimulate apoptosis in both the murine promyelocytic 32D and the human osteosarcoma SAOS2 cell lines when coexpressed with p53. Apoptosis is accompanied by increased transactivation of the cell death-associated BAX gene. This effect is c-myb specific, because B-myb is not able to cooperate with p53 in the induction of BAX transcription and apoptosis. Immunoprecipitation studies and gel shift analysis indicate that c-myb does not directly interact with the BAX promoter or the p53 protein but, rather, cooperates through an indirect mechanism. Consistent with the existence of a functional link between c-myb and p53, we also observed that c-myb represses p53-induced activation of the WAF-1 promoter and induces proliferation of SAOS2 cells growth arrested by p53. These results might contribute to the elucidation of the mechanisms underlying p53-dependent pathways of oncogene-induced apoptosis and provide a further example of DNA-binding independent myb activity.

Animals↗
PubMed1996

Nitric oxide induced poly(ADP-ribose) polymerase cleavage in RAW 264.7 macrophage apoptosis is blocked by Bcl-2.

Endogenously generated or exogenously supplied nitric oxide causes cleavage of poly(ADP-ribose) polymerase (PARP) and apoptotic cell death in RAW 264.7 macrophages. With the use of NO donors such as S-nitrosoglutathione or spermine-NO we established that PARP digestion occurs in parallel with DNA fragmentation, and is preceded by accumulation of the tumor suppressor gene product p53. PARP cleavage in response to lipopolysaccharide and interferon-gamma treatment is prevented by NG-monomethyl-L-arginine, thus proving a NO requirement. Endogenous NO generation, p53 accumulation, and PARP degradation occurred prior to the detection of significant chromatin condensation. In contrast, in stable Bcl-2 transfected cells, NO-initiated PARP cleavage was almost completely blocked. Our data implicate PARP as a proteolytic substrate during NO-mediated apoptotic cell death in RAW 264.7 macrophages and establish Bcl-2 as an efficient signal terminator in this process.

Animals↗
PubMed1996

Integrin activation by R-ras.

Expression of a constitutively active R-ras converted two cell lines that grow in suspension into highly adherent cells. There was little change in cell surface expression of integrins, but attachment to surfaces coated with the integrin ligands was greatly enhanced. Cells transfected with activated R-ras bound integrin ligands from solution with higher affinities and assembled severalfold more fibronectin matrix than control transfectants. Introduction of a dominant negative R-ras into adherent cells reduced the adhesiveness of the cells, indicating that endogenous R-ras can control the ligand-binding activity of integrins. These results provide a mechanism for the modulation of integrin ligand-binding activity as well as novel function for R-ras.

Animals↗
PubMed1996

Apoptosis sensitivity in chronic lymphocytic leukemia is determined by endogenous endonuclease content and relative expression of BCL-2 and BAX.

Therapeutic agents used in the treatment of chronic lymphocytic leukemia (CLL) are capable of inducing apoptosis in some (but not all) patient isolates. It is not yet clear whether cells that are resistant to one agent will also be resistant to others, and the mechanisms contributing to differential apoptosis sensitivity are not known. Here we report that glucocorticoid hormone and a clinically relevant chemotherapy combination (fludarabine plus mitoxantrone) fail to induce apoptosis in four of 24 CLL patient isolates. Apoptosis resistance was associated with elevated BCL-2 and BAX expression. Interestingly, incubation in vitro led to down-regulation of BCL-2 expression in both apoptosis-sensitive and apoptosis-resistant cells, whereas parallel down-regulation of BAX occurred only in the resistant samples. Evaluation of nuclear endonuclease content indicated that all of the apoptosis-sensitive samples contained appreciable levels of activity, whereas the endonuclease was not detected in the four populations of resistant cells. Our results indicate that nuclear endonuclease activity represents an excellent prognostic indicator of CLL apoptosis sensitivity that may be controlled by differential BCL-2 family polypeptide expression and signals from the in vivo microenvironment.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1996

Proapoptotic protein Bax heterodimerizes with Bcl-2 and homodimerizes with Bax via a novel domain (BH3) distinct from BH1 and BH2.

Most members of the Bcl-2 protein family of apoptosis regulating proteins contain two evolutionarily conserved domains, termed BH1 and BH2. Both BH1 and BH2 in the Bcl-2 protein are required for its function as an inhibitor of cell death and for heterodimerization with the proapoptotic protein Bax. In this report, we mapped the region in Bax required for heterodimerization with Bcl-2 and homodimerization with Bax, using yeast two-hybrid and in vitro protein-protein interaction assays. Neither the BH1 nor the BH2 domain of Bax was required for binding to the wild-type Bcl-2 and Bax proteins. Moreover, Bax (deltaBH1) and Bax (deltaBH2) mutant proteins bound efficiently to themselves and each other, further confirming the lack of requirement for BH1 and BH2 for Bax/Bax homodimerization. Bax/Bax homodimerization was not dependent on the inclusion of the NH2-terminal 58 amino acids of the Bax protein in each dimerization partner, unlike Bcl-2/Bcl-2 homodimers which involve head-to-tail interactions between the region of Bcl-2 where BH1 and BH2 resides, and an NH2-terminal domain in Bcl-2 that contains another domain BH4 which is conserved among antiapoptotic members of the Bcl-2 family. Similarly, heterodimerization with Bcl-2 occurred without the NH2-terminal domain of either Bax or Bcl-2, suggesting a tail-to-tail interaction. The essential region in Bax required for both homodimerization with Bax and heterodimerization with Bcl-2 was mapped to residues 59-101. This region in Bax contains a stretch of 15 amino acids that is highly homologous in several members of the Bcl-2 protein family, suggesting the existence of a novel functional domain which we have termed BH3. Deletion of this 15-amino acid region abolished the ability of Bax to dimerize with itself and to heterodimerize with Bcl-2. The findings suggest that the structural features of Bax and Bcl-2 that allow them to participate in homo-and heterodimerization phenomena are markedly different, despite their amino-acid sequence similarity.

Amino Acid Sequence↗
PubMed1996

A cis-acting element in the BCL-2 gene controls expression through translational mechanisms.

The bcl-2 gene becomes activated in many types of human cancers and contributes to neoplastic cell expansion, as well as to resistance to radiation and chemotherapy, by blocking programmed cell death or apoptosis. The expression of this proto-oncogene is regulated at both the transcriptional and post-transcriptional levels. DNA sequence comparisons of human, mouse, rat and chicken bcl-2 cDNAs revealed the presence of an open reading frame (ORF) [correction of (OFR)] located upstream of the normal coding region. Because upstream ORFs (uORFs) have been associated with translational repression, we analysed the functional significance of the 11 amino-acid uORF in the human BCL-2 gene (-119 to -84 bp). Deletion of this uORF from chloramphenicol acetyltransferase (CAT) reporter gene constructs that contained the bcl-2 promoter and entire 5'-untranslated region (5'-UTR), as well as introduction of an A-->T mutation at position -119 bp that destroyed the AUG-initiation codon, significantly increased CAT activity in HeLa, CEM, and other cell lines, without producing a corresponding elevation in CAT mRNA levels. Positioning this uORF, together with its accompanying Kozak sequences, between a heterologous promoter from SV40 and a CAT reporter gene resulted in marked inhibition of CAT protein production without a decrease in CAT mRNA. Mutation of the start codon (ATG-->TTG) of this uORF completely abolished its inhibitory activity, consistent with a translational mechanism. Taken together, these findings suggest that the uORF located within the 5'UTR of the bcl-2 gene is necessary and sufficient for translational regulation of bcl-2 gene expression.

Animals↗
PubMed1996

Antisense oligodeoxynucleotides to bax mRNA promote survival of rat sympathetic neurons in culture.

Previous in vitro studies have shown that the presence of high levels of Bax protein accelerated the rate of cell death following growth factor deprivation and that the ratio of cell death repressor Bcl-2 to cell death effector Bax may determine the susceptibility to apoptosis. Both Bcl-2 and Bax protein expression has been detected in sympathetic neurons in vivo, and overexpression of bcl-2 in cultured sympathetic neurons prevented apoptosis after deprivation of nerve growth factor (NGF). In the present study, we investigated the expression of bax and bcl-2 in primary cultures of sympathetic neurons from rat superior cervical ganglia. Furthermore, we tested the effects of a partially phosphorothioated bax antisense oligodeoxynucleotide (ODN) on the survival of sympathetic neurons in cultures supplied with suboptimal concentrations of NGF (0.5 ng/ml). A constitutive expression of bax mRNA at high levels was detected by reverse transcription and polymerase chain reaction which did not change significantly following NGF reduction or treatment with bax antisense ODN. A decrease in Bcl-2 immunoreactivity was observed by immunocytochemistry in tyrosine hydroxylase-positive neurons when cultured under suboptimal NGF concentrations, whereas Bcl-2 immunolabeled non-neuronal cells were not affected. Maximal number of neurons was obtained in control cultures containing 50 ng/ml of NGF. Few neurons survived in cultures grown in 0.5 ng/ml of NGF for 2 days (12.0 +/- 1.5% of controls, mean +/- SEM). Addition of two control ODNs at 1 microM had no effect on neuronal survival (10.1 +/- 1.2% and 11.0 +/- 1.3%, respectively), while the number of neurons was significantly increased in NGF-reduced cultures treated with a bax antisense ODNs (1 microM) (31.5 +/- 1.9%). Administration of fluorescein-labeled ODNs demonstrated intracellular uptake into cultured neurons. Treatment with bax antisense ODNs caused a significant reduction of Bax protein levels in SCG neurons by 46 +/- 2.6% as assessed by immuno-cytochemistry and digital image analysis. Taken together, our data demonstrate a constitutive expression of bax mRNA in sympathetic neurons suggesting that activation of bax expression may not be required for neuronal cell death after NGF withdrawal. After changing to suboptimal NGF concentrations, the cell-specific reduction in Bcl-2 immunoreactivity preceded morphological signs of degeneration indicating that growth factor starvation may down-regulate neuronal bcl-2 expression. Treatment with bax antisense ODNs indicated that suppression of Bax protein synthesis may promote neuronal survival in the threshold situation of insufficient trophic support.

Animals↗
PubMed1996

Frequent expression of the cell death-inducing gene Bax in Reed-Sternberg cells of Hodgkin's disease.

The expression of a cell death-inducing gene, Bax, was investigated in 52 cases of Hodgkin's disease in parallel with Epstein-Barr virus and was compared with the immunodetection of other apoptosis-regulating proteins, Mcl-1, Bcl-2, and Bcl-x. Bax immunostaining was found in 92% of the cases, among them 28% with a strong signal in more than 75% of the Reed-Sternberg cells. Mcl-1 was positive in 80% of the cases, whereas Bcl-2 and Bcl-x were found in 53% and 88% of the cases, respectively. Of 48 (89%) Bax-positive tumors, 43 were found to express apoptosis-inhibiting proteins such as Mcl-1 or Bcl-2. With the exception of 1 case, all Bax-positive tumors also expressed either Bcl-2, Bcl-x, Mcl-1, or combinations of these anti-apoptotic proteins. No correlation was found between Bax expression and the presence of apoptotic cells as detected by morphology and the in situ 3' OH-DNA end-labeling technique. Our findings show that the apoptosis-inducing gene Bax expression is frequently expressed in Hodgkin's disease, providing a potential explanation for the good chemoresponses generally obtained for patients with this neoplastic disorder.

Apoptosis↗
PubMed1996

Drug-induced apoptosis in B-cell chronic lymphocytic leukemia: relationship between p53 gene mutation and bcl-2/bax proteins in drug resistance.

We investigated the relationship among chemosensitivity to drug-induced apoptosis in vitro, the presence of p53 gene mutations, and the expression of bcl-2 and bax proteins in B-cells from B-cell chronic lymphocytic leukemia (B-CLL) patients. Apoptosis was induced with a camptothecin analogue, 9-amino-20(s)-camptothecin, or a purine analogue, fludarabine. Cell death was monitored by propidium iodide staining and FACS analysis. Drug-induced apoptosis in B-CLL cells was p53-independent. Immunoblot analysis of bcl-2 and bax expression revealed a correlation between drug-induced apoptosis and the ratio of endogenous levels of bcl-2 to bax proteins. B-CLL cells with none to low bcl-2/bax ratios were drug-sensitive as compared to cells with intermediate to high ratios that were drug-resistant (P = 0.015). Prior to drug treatment, bax protein migrated as a single species of 21 kDa. Following drug-induced apoptosis, anti-bax specific protein complexes of 36-42 kDa were up-regulated. Using two-dimensional gel electrophoresis, bax complexes were disrupted under reducing conditions to reveal homo- and heterodimers of 18 and 21 kDa suggesting that disulfide interactions were required for complex formation. The de novo appearance of the 18 kDa anti-bax specific protein together with its increased expression in drug-sensitive B-CLL B-cells undergoing cell death suggests a role for this protein in the regulation of apoptosis.

Apoptosis↗
PubMed1996

A bcl-2 transgene expressed in hepatocytes protects mice from fulminant liver destruction but not from rapid death induced by anti-Fas antibody injection.

Stimulation of the Fas (APO-1, CD95) receptor, which is present on a variety of cells, usually triggers a process of programmed cell death. Systemic injection of anti-Fas antibody into mice leads to fulminant liver destruction resulting from massive hepatocyte apoptosis, and to rapid death. Hepatocytes bear Fas but do not express Bcl-2, a protein that plays, in a number of conditions, a protective role against apoptosis. We have generated mice whose liver expresses Bcl-2 as the result of bcl-2 transgene placed under the control of the hepatocyte-specific alpha1-anti-trypsin gene promoter, but is otherwise not distinguishable from that of normal mice. These mice display a marked to almost total resistance to liver damage induced by anti-Fas antibody injection. This protective effect of Bcl-2 occurs in the absence of significant variations, in the stimulated livers, in the level of expression of other proteins also involved in resistance or sensitivity to apoptosis, namely Bcl-x, Bax, Bad, Bak, and p53. Mice with protected livers, however, die almost as rapidly as normal mice, which indicates that acute lethality results from stimulation of Fas receptors present on other target organs or cells.

Animals↗
PubMed1996

A1, a Bcl-2 family member, prolongs cell survival and permits myeloid differentiation.

A1, a bcl-2 family member, has been identified as a hematopoietic-specific, early inducible gene. In this study it is shown that stable transfection of A1 into an interleukin-3 (IL-3)-dependent myeloid precursor cell line, 32D c13, leads to a retardation of IL-3 withdrawal-induced cell death similar to that observed with transfection of bcl-2. However, unlike bcl-2. A1 expression permits the accumulation of differentiated myeloid cells both before and after IL-3 withdrawal. Total cell accumulation, on the other hand, is considerably greater after IL-3 deprivation in the bcl-2 transfectant than in A1-expressing cells. Cells cotransfected with the two genes behave similarly to cells singly transfected with bcl-2, except that viability following IL-3 withdrawal is somewhat further enhanced. These results suggest that these two proteins have distinct roles that may be related to the divergent regulation of their expression during myeloid differentiation.

Animals↗
PubMed1996

Association between cisplatin resistance and mutation of p53 gene and reduced bax expression in ovarian carcinoma cell systems.

P53 status may be a determinant of chemosensitivity of tumor cells; however, its involvement in cellular resistance to cisplatin remains uncertain. To investigate the relationships between p53 and the development of resistance to cisplatin, the p53 gene status was studied in ovarian carcinoma cell systems which included two cisplatin-resistant variants (IGROV-1/Pt 0.5 and IGROV-1/Pt 1) selected in vitro after prolonged drug exposure of the cisplatin-sensitive parental IGROV-1 cell line. IGROV-1/Pt 0.5 and IGROV-1/Pt 1 cell lines exhibited a degree of resistance of approximately 6 and 14, respectively, following 96-h exposure to the drug and were cross-resistant to other DNA-damaging agents (ionizing radiation and melphalan). Resistance to cisplatin paralleled a reduced cell susceptibility to cisplatin-induced apoptosis. DNA single-strand conformation polymorphism analysis of exons 5-9 demonstrated the presence of two mutants alleles at exon 8 in the two resistant cell lines, in contrast to the parental IGROV-1 cell line which exhibited the wild-type p53 gene. Direct DNA sequencing revealed that the mutations consist of two nucleotide changes in the DNA-binding domain at codons 270 (T/A) and 282 (C/T). The consecutive levels of p53 protein were lower in IGROV-1 than in IGROV-1/Pt cells. Following exposure to ionizing radiation or cisplatin, accumulation of the p53 protein was markedly enhanced only in the sensitive cells. Concomitantly, the expression of WAF-1 protein was strongly induced in the parental IGROV-1 cells, whereas WAF-1 protein remained undetectable in the IGROV-1/Pt 1 subline after DNA-damaging treatment. Consistent with this finding is the observation that ionizing radiation caused a different pattern of cell cycle perturbation in sensitive and resistant cells. Northern blot analysis demonstrated a marked reduction in bax mRNA levels in IGROV-1/Pt 1 cisplatin-resistant cells. Cotransfection assays with wild-type or mutant p53 expression plasmids and a reporter gene plasmid that utilized the bax gene promoter to drive transcription of chloramphenicol acetyltransferase were consistent with the role of p53 in regulation of bax expression in these cells. Taken together, these observations support a role for mutations of the p53 gene in the development of cisplatin resistance in ovarian cancer as a consequence of loss of the ability of p53 to transactivate bax, an apoptosis-inducing gene.

Apoptosis↗