Overexpression of BLM promotes DNA damage and increased sensitivity to platinum salts in triple-negative breast and serous ovarian cancers.

Background: Platinum-based therapy is an effective treatment for a subset of triple-negative breast cancer and ovarian cancer patients. In order to increase response rate and decrease unnecessary use, robust biomarkers that predict response to therapy are needed. Patients and methods: We performed an integrated genomic approach combining differential analysis of gene expression and DNA copy number in sensitive compared with resistant triple-negative breast cancers in two independent neoadjuvant cisplatin-treated cohorts. Functional relevance of significant hits was investigated in vitro by overexpression, knockdown and targeted inhibitor treatment. Results: We identified two genes, the Bloom helicase (BLM) and Fanconi anemia complementation group I (FANCI), that have both increased DNA copy number and gene expression in the platinum-sensitive cases. Increased level of expression of these two genes was also associated with platinum but not with taxane response in ovarian cancer. As a functional validation, we found that overexpression of BLM promotes DNA damage and induces sensitivity to cisplatin but has no effect on paclitaxel sensitivity. Conclusions: A biomarker based on the expression levels of the BLM and FANCI genes is a potential predictor of platinum sensitivity in triple-negative breast cancer and ovarian cancer.

BRCA2 mutations in primary breast and ovarian cancers.

The second hereditary breast cancer gene, BRCA2, was recently isolated. Germline mutations of this gene predispose carriers to breast cancer, and, to a lesser extent, ovarian cancer. Loss of heterozygosity (LOH) at the BRCA2 locus has been observed in 30-40% of sporadic breast and ovarian tumours, implying that BRCA2 may act as a tumour suppressor gene in a proportion of sporadic cases. To define the role of BRCA2 in sporadic breast and ovarian cancer, we screened the entire gene for mutations using a combination of techniques in 70 primary breast carcinomas and in 55 primary epithelial ovarian carcinomas. Our analysis revealed alterations in 2/70 breast tumours and none of the ovarian carcinomas. One alteration found in the breast cancers was a 2-basepair (bp) deletion (4710delAG) which was subsequently shown to be a germline mutation, the other was a somatic missense mutation (Asp3095Glu) of unknown significance. Our results suggest that BRCA2 is a very infrequent target for somatic inactivation in breast and ovarian carcinomas, similar to the results obtained for BRCA1.

Immunotherapy of a murine leukemia virus-infected, chemically induced murine sarcoma with antiviral antibodies.

Many murine tumor models associated with murine leukemia virus(es) (MuLV) have been successfully treated by passive administration of antiviral antibodies. There is a large body of virus-negative tumors, however, which are lowly antigenic and thus refractory to such approaches. Therefore, an investigation was done for determination of whether such tumors could be rendered susceptible to passive serum therapy by introduction of MuLV antigens onto the tumor cell surface. For this purpose a 3-methylcholanthrene-induced fibro-sarcoma from inbred C57BL/6J mice was chosen. Following infection of the tumor in vitro with Friend murine leukemia virus (F-MuLV), the tumor was found to be susceptible to treatment with a high-titered heterologous anti-F-MuLV gp71 antiserum. The specificity of the treatment was determined by conduction of the therapy on the uninfected parental tumor, in which case there was no effect. In addition, therapy could be initiated at time points when demonstrable tumors were present and successfully treated animals were resistant to rechallenge with the infected tumor. Thus conversion of a lowly antigenic virus-negative tumor to an MuLV-positive antigenic tumor rendered such growths susceptible to immunologic management.

In vivo antigenic modification of tumor cells. III. Metastatic thymic lymphoma specifically infected by thymotropic retrovirus.

Tissue distribution of radiation leukemia virus (RadLV) was examined after its inoculation into normal C57BL/6J (B6) mice, B6 mice bearing a transplantable, non-virus-producing thymic lymphoma (RL12-NP), and B6 mice bearing a transplanted non-virus producing, Harvey murine sarcoma virus-transformed fibrosarcoma. Virus expression was determined by competition radioimmunoassay for murine leukemia virus (MuLV) p30 (predominant group-reactive antigen of MuLV) and for RadLV p12 (a highly type-specific MuLV polypeptide) and by membrane immunofluorescence for cell surface gp71 (predominant envelope glycoprotein of MuLV). Normal adult B6 mice were given three sequential iv injections of RadLV and were examined several times up to 200 days later for the appearance of neoplastic disease or expression of virion antigens. No clinical abnormalities were noted, and animals remained healthy for greater than 200 days. Significant levels of MuLV p30 and RadLV p12 were detected only in the thymuses. Organs and tumors from RL12-NP-inoculated animals contained low or nondetectable levels of virion antigens. Inoculation of mice with RL12-Rad, a cell line derived by in vitro infection of RL12-NP cells with RadLV, produced widespread, discrete metastatic tumors and infiltrated the lymphoid organs of B6 mice in a pattern identical to that observed after administration of RL12-NP cells. Lymphoid organs of RL12-Rad-inoculated animals expressed variable levels of virion antigens reflecting differences in the extent of tumor cell infiltration as opposed to virus spread from tumor to host cells. Administration of infectious RadLV systemically into RL12-NP tumor-bearing animals converted these tumors to viron antigen expressors with levels in superinfected tumors equivalent to those found in RL12-Rad-induced tumors. Infection was highly selective, and host tissues were minimally contaminated by the inoculated virus. Part of this selectivity was explained by the thymotropic property of RadLV. A rapidly dividing murine fibrosarcoma was not infected by RadLV, but this same non-virus-expressing tumor could be infected by common fibrotropic MuLV isolates.

In vivo antigenic modification of tumor cells. II. Distribution of virus in sarcoma-bearing mice.

Murine leukemia viruses were previously demonstrated to be able to infect efficiently non-virus-expressing tumors in vivo. In the present study the infectivity and tissue distribution of Friend murine leukemia virus (F-MuLV) in normal and tumor-bearing C57BL/6J (B6) mice were examined. Two syngeneic fibrosarcoma-inducing cell lines were used: Cells from a 3-methylcholanthrene-induced fibrosarcoma syngeneic to B6 mice (MCA-FS) and cells from a Harvey murine sarcoma virus-transformed, nonproducer sarcoma syngeneic to B6 mice (H-NP) were described in the preceding study. Both cell lines lacked ecotropic viral expression. F-MuLV produced in vitro was rarely able to infect normal adult B6 tissue in vivo and lacked pathogenic potential. Adult animals receiving F-MuLV remained clinically normal during 20 months of follow-up and had no detectable viremia, although some had persistently infected thymuses and long bones. In animals receiving a single dose of F-MuLV given to superinfect either the MCA-FS or the H-NP induced tumors, virion antigens were found only in tumor tissue and not in the normal host organs studied. Infectious virus was abundant in tumors; occasionally, it was found in thymuses and long bones of animals bearing superinfected H-NP tumors but rarely in other organs. Localization of F-MuLV in MCA-FS tumors appeared to be more selective with rare contamination of host organs. The presence of a rescuable sarcoma genome in H-NP may explain the discrepancy between MCA-FS and H-NP tumors. The possibility of increasing the efficiency and selectivity of infection as well as the therapeutic application of this technique are discussed.

In vivo antigenic modification of tumor cells. I. Introduction of murine leukemia virus antigens on non-virus-producing murine sarcomas.

Murine oncovirus antigens represent excellent targets for immune recognition, and virus-associated tumors are generally susceptible to various immunotherapy protocols. Virus-negative tumors, however, are nonimmunogenic and refractory to immunologic control. Therefore, the feasibility of the introduction of antigens onto non-virus-expressing tumors in situ in inbred C57BL/6J mice by systemic administration of nononcogenic murine retroviruses was investigated. Two classes of murine fibrosarcomas were studied: a 3-methylcholanthrene-induced fibrosarcoma syngeneic to C57BL/6 mice (MCA-FS) and a Harvey murine sarcoma virus-transformed, nonproducer fibrosarcoma syngeneic to C57BL/6 mice (H-NP). Both were found to be devoid of infectious ecotropic murine leukemia virus (MuLV) or MuLV antigens. A single dose of Friend murine leukemia virus (F-MuLV) was used to superinfect MCA-FS- and H-NP-induced tumors in vivo and converted these tumors to a highly productive, virus-positive state. In vivo superinfected tumors were indistinguishable from their preinfected counterparts by competition radioimmunoassays for the virion's major envelope glycoprotein, gp71, and its group-specific antigen, p30, and by assays for infectious virus. Analysis of virus from tumor extracts proved that the antigenic specificity of the superinfected tumor was provided by F-MuLV administered systemically to the animals. Finally, an immunoperoxidase technique, applied to tumor cross sections, demonstrated the uniform appearance of viral antigens in the superinfected tumors.

Overexpression of the protein tyrosine phosphatase PTP1B in human breast cancer: association with p185c-erbB-2 protein expression.

BACKGROUND: The p185c-erbB-2 growth factor receptor protein tyrosine kinase (PTK) is overexpressed in one third of human breast cancer patients and indicates a poor prognosis in these patients. Protein tyrosine phosphatases (PTPs) may balance PTK activity as part of normal growth-regulation pathways. PTP1B is an intracellular PTP that is involved in linkage between signal transduction pathways and may interface with inappropriate PTK activity in transformed cells. PURPOSE: The aim of this study was to determine if PTP1B is overexpressed in human mammary tumors and to determine if such overexpression is associated with the overexpression of the p185c-erbB-2 receptor PTK. METHODS: Our samples were frozen sections from 29 human mammary tumors (19 pure infiltrating, two pure intraductal, and eight combined intraductal and infiltrating) and nine sections from normal breast tissue. The sections were immunohistochemically stained for PTP1B and p185c-erbB-2, and the results were analyzed statistically for association between overexpression of the two proteins. Northern blot analysis was used to assess if PTP1B overexpression was coincident with increased transcription of the PTP1B gene. RESULTS: Overexpression of the PTP1B protein was observed in 72.4% of the tumor sections compared with normal epithelium, with maximal expression occurring in 37.9% of the tumors. All of the tumor subtypes, including the pure intraductal lesions, overexpressed PTP1B. Statistical analyses demonstrated a significant association between PTP1B overexpression and breast cancer (P < .038) and between the overexpression of PTP1B and the overexpression of p185c-erbB-2 (P < .006). PTP1B messenger RNA steady-state transcription was consistently increased in tumors versus normal tissues. CONCLUSIONS: PTP1B overexpression is a common phenotypic manifestation in human breast cancers and is associated with over-expression of p185c-erbB-2. Steady-state PTP1B transcription is increased in tumor tissue. IMPLICATIONS: Further studies are needed to determine if PTP1B overexpression balances or augments PTK activity. PTP1B overexpression might also be evaluated as a clinical prognostic factor in human breast cancers.

Maintenance of p53 alterations throughout breast cancer progression.

Overexpression of the nuclear phosphoprotein p53 is one of the most common abnormalities in primary human cancer and appears to be due to point mutation within a highly conserved region of the p53 gene which then encodes for a mutant, more stable protein. In this study different stages of breast cancer progression were examined, from in situ to metastatic disease, to determine at what stage mutational activation occurs and whether it is maintained during tumor progression. Two (13%) of 15 pure intraductal tumors expressed high levels of p53 in all malignant epithelial cells. Sequencing of p53 mRNA from one of these tumors demonstrated a nucleotide substitution altering the amino acid composition of the protein. Six (17%) of 35 specimens which contained both in situ and invasive disease expressed high levels of p53. All malignant epithelial cells in these 6 cases stained positively and in no specimen did one component express different levels of the protein than the other growth phase. Sequence analysis of a tissue with significant amounts of both in situ and invasive disease revealed only a single point mutation, without evidence of wild-type nucleotide at the site of substitution, suggesting that p53 mRNA from each component of the tumor contained the same nucleotide substitution. Eleven (50%) of 22 pairs of primary tumors and their lymph node metastases expressed elevated levels of p53, and in each case, expression levels were identical in the primary and secondary sites. Identical mutations were found in the p53 mRNA from two paired primary and metastatic sites. Therefore, mutation within a highly conserved region of the p53 gene leading to overexpression of the protein product can occur in the earliest recognized phase of breast cancer and this alteration is maintained during progression from intraductal to infiltrating carcinoma. Mutations are also conserved during the process of metastatic spread.

Loss of heterozygosity for genes on 11p and the clinical course of patients with lung carcinoma.

Forty-five primary human lung carcinomas were evaluated for the loss of heterozygosity for genes on the short end of chromosome 11. Of 40 evaluable heterozygous cases, loss of the 11p genes c-H-ras and insulin was documented in nine cases (22%). The clinical parameters investigated for each patient included the disease stage at presentation, the presence of metastatic disease in either bronchial or mediastinal lymph nodes, and the presence of positive parietal pleural margins in the surgically resected specimen. There were no differences found with respect to these indicators when patients exhibiting the loss of heterozygosity were compared with those who did not have such genetic loss. In addition, when the clinical courses of the two patient groups were compared, there was no difference in survival. We conclude that the loss of heterozygosity for c-H-ras and insulin on 11p is a common finding in primary non-small cell human lung carcinomas but does not confer a more aggressive phenotype on these tumors. Although this genetic lesion may be important in the initial transformation of the cells to carcinoma, the available data for lung carcinoma are insufficient to prove causality.

A prognostic model of recurrence and death in stage I non-small cell lung cancer utilizing presentation, histopathology, and oncoprotein expression.

In order to construct a multivariate model for predicting early recurrence and cancer death for patients with stage I non-small cell lung cancer, 271 consecutive patients (mean age, 63 +/- 8 years) who were diagnosed, treated, and followed at one institution were studied. All patients were clinical stage I with head and chest/abdominal computed tomograms and radionuclide bone scans without evidence of metastatic disease. Pathological material after resection was reviewed to verify histological staging. Follow-up documented the time and location of any recurrence, was a median 56 months in duration, and was complete in all cases. Data recorded included age, sex, smoking history, presenting symptoms, pathological description, and oncoprotein staining for erbB-2 (HER-2/neu), p53, and KI-67 proliferation protein. Immunohistochemistry of oncogene expression was performed on two separate archived paraffin tumor blocks for each patient, with normal lung as control. All analyses were blinded and included Kaplan-Meier survival estimates with Cox proportional hazards regression modeling. Data, including immunohistochemistry, were complete for all 271 patients. Actual 5-year survival was 63% and actuarial 10-year survival was 58%. Significant univariate predictors (P < 0.05) of early recurrence and cancer-death were: male sex; the presence of symptoms; chest pain; type of cough; hemoptysis; tumor size > 3 cm diameter (T2); poor differentiation; vascular invasion; erbB-2 expression; p53 expression; and a higher KI-67 proliferation index (> 5%). An additive oncogene expression curve demonstrated a 5-year survival of 72% for 136 patients without p53 or erbB-2, 58% for 108 patients who expressed either oncogene, and 38% for 27 who expressed both (P < 0.001).(ABSTRACT TRUNCATED AT 250 WORDS)

Increased erbB-2 gene copies and expression in multiple stages of breast cancer.

In order to examine the role of the erbB-2 oncogene in human breast cancer, gene amplification and expression were examined in multiple stages of tumor progression. Gene amplification ranging from 2-fold to 32-fold was found in 30 (29%) of 130 cases analyzed. Expression of the receptor-like gene product was determined by a combination of Western immunoblotting and immunohistochemistry. In each case of gene amplification, there was high level overexpression (+ + +) of the protein product. In an additional 29 of 111 cases in which expression was studied (26%), there was moderate level overexpression (+ +) of erbB-2 in the absence of gene amplification. Amplification and overexpression of the erbB-2 gene were found in early clinical stages of breast cancer as well as in more advanced cases. In 23 patients, gene number and level of gene expression were equivalent in the primary tumor site compared with single or multiple metastatic sites in regional lymph nodes. Using a combination of immunohistochemistry and in situ cytohybridization, high (+ + +) and moderate (+ +) level overexpression were homogeneously present in all malignant epithelial cells within histological sections of both primary and metastatic tumor. The intraductal component of carcinoma was identified in sections from 16 invasive primary tumors. erbB-2 gene expression in the intraductal lesions was equivalent to or exceeded expression in the infiltrating components of these tumors. Because erbB-2 alterations are (a) present in all clinical stages, (b) maintained during metastatic spread, (c) homogeneously present throughout tumor sections, and (d) present in the in situ as well as infiltrating component, we conclude that these alterations are selected for early and may be important in the initiation of certain mammary cancer.

Detection of frequent allelic loss on proximal chromosome 17q in sporadic breast carcinoma using microsatellite length polymorphisms.

Analyses of losses of heterozygosity and linkage studies have implicated a gene(s) on chromosome 17q in the genesis of sporadic and early-onset familial breast carcinomas, respectively. To define the critical region of 17q, we examined DNAs from a series of 20 sporadic breast carcinomas and corresponding blood samples for allelic losses of chromosome 17q using microsatellite length polymorphisms. With these highly informative markers (average heterozygosity, 0.73), we observed frequent deletions of 17q at several loci. We found that D17S250 was deleted in 50% (7 of 14), THRA1 in 79% (11 of 14), D17S579 in 59% (11 of 19), NME1 in 29% (5 of 17), MPO in 36% (4 of 11), and GH in 25% (4 of 16) in the tumor set examined. A common region of deletion was found that was flanked by D17S250 to D15S579. These markers have recently been localized to a 6-cM interval of proximal chromosome 17q in bands 17q11.2-q21 and map within the region of the early-onset familial breast cancer locus, implying that the same gene or genes may be involved in both sporadic and familial breast tumors. Thyroid hormone receptor alpha and retinoic acid receptor alpha are two potential candidate genes in this region.

Cell cycle control of BRCA2.

Identifying the conditions and kinetics of the induction of BRCA2 gene expression may implicate roles for the function of the tumor suppressor gene. In this study, expression of BRCA2 mRNA is shown to be regulated by the cell cycle and associated with proliferation in normal and tumor-derived breast epithelial cells. Cells arrested in G(0) or early G1 contained low levels of BRCA2 mRNA. After release into a proliferating state, cells produced maximum levels of BRCA2 mRNA in late G1 and the S-phase. Similar cell cycle control of BRCA2 was observed in fractions of exponentially growing cells isolated by centrifugal elutriation. Expression of BRCA2 was shown to be independent of bulk DNA synthesis. In addition, the kinetics of BRCA2 mRNA up-regulation appeared to be similar to those of BRCA1, suggesting that the two genes could be commonly controlled. These results imply that these two tumor suppressor genes are utilized during growth and may have a protective role in cellular proliferation.

Morphological and biochemical properties of a new human breast cancer cell line.

DU4475 is a new human breast cell line derived from a cutaneous metastatic nodule from a patient with advanced breast cancer. It has been in continuous culture for more than 1 year and has survived 140 subcultivations. The cells grow in suspension, displaying clusters and free-floating aggregates with serpentine-like outgrowths. The cell line is hypotetraploid with modal chromosome numbers between 86 and 93 and possesses five to six distinctive marker chromosomes. The cells grow readily in athymic nude mice and produce tumors from which cells can be reintroduced to culture in vitro.

Mutation analysis of the THRA1 gene in breast cancer: deletion/fusion of the gene to a novel sequence on 17q in the BT474 cell line.

We have previously described a common region of deletion and allele loss on chromosome 17q in sporadic breast cancers that is likely to contain a tumor suppressor gene. The region, mapped to 17q12-q21, was bordered by D17S250 and D17S579 on the centromeric and telomeric sides, respectively. This deletion region overlaps the BRCA1 locus, which predisposes to familial breast and ovarian cancer. The most frequent loss of heterozygosity was observed at the thyroid hormone receptor alpha (THRA1) locus. Southern analysis revealed a rearrangement of THRA1 in the BT474 breast cancer cell line. This rearrangement represented a deletion of exons 8-10 of one THRA1 allele that was also coamplified with ERBB2. Northern blots showed two mutant transcripts in BT474 cells. Analysis of the mutant transcripts revealed fusion of the THRA1 exon 7 by splicing to a novel sequence designated BTR for "BT474 transcribed rearrangement." BTR was found to be highly conserved and mapped to 17q. The deletion in BT474 cells spans the entire BRCA1 region. To search for additional mutations in the THRA1 gene, all nine protein-encoding exons of THRA1 were examined for point mutations via single strand conformation analysis in a series of primary breast tumors, breast cancer cell lines, and lymphoblastoid cell lines derived from the youngest affected members of several German breast cancer families. No point mutations were detected, including the unrearranged THRA1 allele in BT474. We have thus excluded THRA1 as a commonly mutated sporadic breast cancer tumor suppressor gene and as the BRCA1 gene.

Overexpression and mutation of p53 in epithelial ovarian cancer.

We examined p53 expression in 107 epithelial ovarian cancers with immunohistochemical techniques using monoclonal antibody PAb1801. High level expression of nuclear p53 protein was detected in the malignant epithelium in 54 (50%) of these cancers. Expression of p53 protein was undetectable in 13 benign gynecological tissues. p53 mRNA from three cancers that overexpressed the protein were sequenced and point mutations which altered the coding sequence of the highly conserved region of the gene were found in each case. Three cancers with undetectable protein levels also were sequenced and were found to be wild-type through the same region of the gene. As in other cancers, overexpression of the p53 protein in ovarian cancer appears to correlate closely with the presence of mutation in the p53 gene. p53 overexpression did not correlate with stage, histological grade, or the ability to perform optimal cytoreductive surgery. A significant correlation (P = 0.04) was observed between p53 overexpression and aneuploidy in advanced stage (III/IV) disease. There was no significant relationship between overall survival and p53 expression. Since mutation and overexpression of p53 are common in epithelial ovarian cancers, further studies are warranted to clarify the role of p53 in ovarian tumorigenesis.

Overexpression and mutation of p53 in endometrial carcinoma.

Immunohistochemical staining for the p53 protein was performed in 107 snap frozen primary endometrial adenocarcinomas and 15 benign uterine tissues using monoclonal antibody PAb1801. No staining was seen in benign samples, whereas intense nuclear staining of cancer cells consistent with overexpression of the p53 protein was observed in 22 of 107 cancers (21%). p53 overexpression was more frequent in advanced (Stage III/IV) cancers (41%) than in early (Stage I/II) cancers (9%) (P less than 0.001), and also was associated with nonendometrioid histology (P = 0.008), positive peritoneal cytology (P = 0.01), extrauterine metastases (P = 0.003), and negative progesterone receptor status (P = 0.04). To confirm the relationship between p53 overexpression and mutation, p53 mRNA from 8 cancers was reverse transcribed and amplified using the polymerase chain reaction. DNA sequencing revealed point mutations in each of the 5 cancers that overexpressed p53, whereas the wild-type sequence was found in 3 cancers that did not overexpress the protein. Each of the 5 mutations resulted in an amino acid substitution in a highly conserved region of the p53 gene where mutations have been found in other cancers. Further studies are warranted to determine whether the association between p53 overexpression and advanced stage disease is due to accumulation of genetic lesions during tumor progression or whether p53 alterations confer a more virulent phenotype.

Overexpression of erbB-2 or EGF receptor proteins present in early stage mammary carcinoma is detected simultaneously in matched primary tumors and regional metastases.

Membrane protein levels of erbB-2 and epidermal growth factor (EGF) receptor as well as gene aberrations affecting these proto-oncogenes in human mammary cancer were determined in primary and metastatic lesions. Among 57 patients erbB-2 gene amplification was detected in 11 tumors (19%). In 10 of these patients where expression levels could be assayed gene amplification was associated with a high level of erbB-2 protein. In contrast, EGF receptor gene amplification with over-expression of the protein product was observed in 2 tumors (4%). In addition, 14 out of 53 (26%) primary tumors exhibited moderately increased erbB-2 protein levels in the absence of gene amplification. Similar aberrations resulting in overexpression of EGF receptor protein without detectable gene amplification were associated with 2 tumors (4%) among 47 patients analyzed. In 7 patients, expression level and gene copy numbers of erbB-2 or EGF receptor were similarly altered in the primary tumor and metastatic lesions derived from the same patient. Concordance of increased receptor gene expression in primary and metastatic lesions combined with the observation that such alterations are detectable as early as stage I and II mammary tumors, suggests that overexpression of erbB protooncogene family members can develop early in breast cancer and is maintained during tumor progression. Comparison of erbB-2 overexpression with clinical disease parameters revealed a correlation of this alteration with inflammatory mammary carcinoma (P = 0.042) implying an association of elevated erbB-2 protein levels with enhanced malignancy of the tumor cell in vivo.

Expression of p53 in human neuroblastoma- and neuroepithelioma-derived cell lines.

Overexpression of the nuclear phosphoprotein p53 has been detected in many different transformed human cell lines and primary adult tumors. Elevated steady-state levels of p53 appear to be the result of an increase in the stability of the protein and, in adult cancers, high levels of the protein are associated with mutation of the p53 gene. In this study, overexpression of p53 was detected in 4 out of 5 human neuroblastoma-derived cell lines. The protein expressed by each of these four lines had a significantly prolonged half-life relative to the p53 protein in immortalized rodent fibroblasts and normal bovine adrenal medullary cells. However, no mutations were detected in the highly conserved regions of the p53 gene in these four neuroblastoma lines and the protein being expressed was not recognized by the mutant-specific anti-p53 monoclonal antibody, PAb 240. Upon retinoic acid-induced differentiation of the LA-N-5 neuroblastoma cell line, the level of p53 protein declined, as did the level of p53 mRNA, but the half-life of the protein remained unchanged. The high level of protein observed in the undifferentiated cell lines appears to result from expression of a stable wild-type p53 protein and increased transcription. In contrast, p53 protein was undetectable in two neuroepithelioma-derived cell lines; the p53 gene in one of these lines contained a nonsense mutation, while the other transcribed truncated p53 mRNA.

Dominance of wild-type p53-mediated transcriptional activation in breast epithelial cells.

The p53 gene is a recessive oncogene whose loss of function can result in cell transformation. Approximately 25% of human breast cancers contain missense mutations in one p53 allele, leading to inactivation of the mutated protein. In almost all of these cases, the wild-type allele is also lost. However, it remains uncertain whether mutant p53 acts in a dominant negative fashion over the wild-type protein. Two parameters of p53 function, transcriptional activation and transcriptional repression, were studied under a variety of experimental conditions within malignant and normal breast epithelial cells. Transient transfection of DNA encoding wild-type p53 was able to transactivate p53-responsive promoters. Wild-type p53 functioned equally well in malignant cells which harbored an endogenous mutation in p53, in malignant cells containing normal p53 and in normal mammary epithelial cells. Co-transfection of cDNAs encoding mutant p53 proteins were unable to inhibit the ability of wild-type p53 to transactivate the reporter constructs. Repression of viral promoters by normal p53 protein was not inhibited by endogenous or co-transfected mutant p53 -proteins. Finally, the p53 regulated gene WAF1/CIP1/p21 was induced following gamma irradiation in normal mammary cells, containing endogenous wild-type p53 and in the same cells transfected with mutant p53 genes. From these experiments we conclude that mutant p53 proteins do not inactivate the transactivating (or repressing) function of a co-expressed normal p53 protein in these cells implying that complete loss of wild-type p53 is required to eliminate these functions in breast epithelium.