Loss Of P53 Gene Research Articles

Establishment of a p53 Null Murine Oral Carcinoma Cell Line and the Identification of Genetic Alterations Associated with This Carcinoma.

Head and neck squamous cell carcinoma (HNSCC), including oral squamous cell carcinoma (OSCC), ranks sixth in cancer incidence worldwide. To generate OSCC cells lines from human or murine tumors, greatly facilitates investigations into OSCC. This study describes the establishing of a mouse palatal carcinoma cell line (designated MPC-1) from a spontaneous tumor present in a heterozygous p53 gene loss C57BL/6 mouse. A MPC-1-GFP cell subclone was then generated by lentivirus infection resulting in stable expression of green fluorescent protein. Assays indicated that MPC-1 was a p53 null polygonal cell that was positive for keratinocyte markers; it also expressed vimentin and showed a loss of E-cadherin expression. Despite that MPC-1 having strong proliferation and colony formation capabilities, the potential for anchorage independent growth and tumorigenesis was almost absent. Like other murine MOC-L and MTCQ cell line series we have previously established, MPC-1 also expresses a range of stemness markers, various oncogenic proteins, and a number of immune checkpoint proteins at high levels. However, the synergistic effects of the CDK4/6 inhibitor palbociclib on other therapeutic drugs were not observed with MPC-1. Whole exon sequencing revealed that there were high rates of non-synonymous mutations in MPC-1 affecting various genes, including Akap9, Arap2, Cdh11, Hjurp, Mroh2a, Muc4, Muc6, Sp110, and Sp140, which are similar to that the mutations present in a panel of chemical carcinogenesis-related murine tongue carcinoma cell lines. Analysis has highlighted the dis-regulation of Akap9, Cdh11, Muc4, Sp110, and Sp140 in human HNSCC as indicated by the TCGA and GEO OSCC databases. Sp140 expression has also been associated with patient survival. This study describes the establishment and characterization of the MPC-1 cell line and this new cell model should help to advance genetic research into oral cancer.

A Glance of p53 Functions in Brain Development, Neural Stem Cells, and Brain Cancer.

p53 is one of the most intensively studied tumor suppressors. It transcriptionally regulates a broad range of genes to modulate a series of cellular events, including DNA damage repair, cell cycle arrest, senescence, apoptosis, ferroptosis, autophagy, and metabolic remodeling, which are fundamental for both development and cancer. This review discusses the role of p53 in brain development, neural stem cell regulation and the mechanisms of inactivating p53 in gliomas. p53 null or p53 mutant mice show female biased exencephaly, potentially due to X chromosome inactivation failure and/or hormone-related gene expression. Oxidative cellular status, increased PI3K/Akt signaling, elevated ID1, and metabolism are all implicated in p53-loss induced neurogenesis. However, p53 has also been shown to promote neuronal differentiation. In addition, p53 mutations are frequently identified in brain tumors, especially glioblastomas. Mechanisms underlying p53 inactivation in brain tumor cells include disruption of p53 protein stability, gene expression and transactivation potential as well as p53 gene loss or mutation. Loss of p53 function and gain-of-function of mutant p53 are both implicated in brain development and tumor genesis. Further understanding of the role of p53 in the brain may provide therapeutic insights for brain developmental syndromes and cancer.

Loss of P16 in Esophageal Adenocarcinoma Detected by Fluorescence in situ Hybridization and Immunohistochemistry

Abstract Molecular biology of esophageal adenocarcinoma (EAC) is not fully elucidated. The aim of this study was to assess the expression of cycle regulator and tumor suppressor p16 in esophageal adenocarcinoma. The expression of p16 at protein and gene level was investigated using immunohistochemistry and fluorescence in situ hybridization in thirteen EAC specimens obtained by endoscopic biopsies and surgical resections. The mean age of enrolled patients was 62 years and a male predominance was observed. Loss of p16 protein expression was detected in 77% of the cases and loss of p16 gene was found in 69% of cases as hemizygous deletion was the most common. Significant correlation was found between the absence of p16 protein expression and p16 allelic loss. Cell cycle disturbances seem to play role in the EAC carcinogenesis and probably p16 gene deletions are connected with the loss of p16 protein expression.

Characterization of the new human pleomorphic undifferentiated sarcoma TP53-null cell line mfh-val2.

Pleomorphic undifferentiated sarcoma (PUS), also called malignant fibrous histiocytoma, is a soft tissue sarcoma which occurs predominantly in the extremities. Its origin is a poorly defined mesenchymal cell, which derives to histiocytic and fibroblastic cells. The patient, a 58year-old man, presented a lesion located in the forearm composed by spindle cells and multinucleated giant cells, which expressed vimentin and adopted a histological pattern formed by irregular-swirling fascicles. Cells were cultured in vitro and a new cell line was established. We characterized this new cell line by histological analyses, cytogenetics (using G-bands and spectral karyotype technique) and cytometric analyses. Cells were grown in culture for more than 100 passages. They had elongated or polygonal morphology. The cells presented a saturation rate of 70,980 cells/cm2, a plating efficiency of 21.5% and a mitotic index of 21 mitoses per field. The cell line was tumorigenic in nude mice. The ploidy study using flow cytometry revealed an aneuploid peak with a DNA index of 1.43. A side population was detected, demonstrating the presence of stem and progenitor cells. Cytogenetics showed a hypotriploid range with many clonal unbalanced rearrangements. Loss of p53 gene was evidenced by MLPA. We describe, for the first time, the characterization of a new human PUS TP53-null cell line called mfh-val2. Mfh-val2 presents a wide number of applications as a TP53-null cell line and a great interest in order to characterize genetic alterations influencing the oncogenesis or progression of PUS and to advance in the biological investigation of this tumor.

Mutant p53-R273H gains new function in sustained activation of EGFR signaling via suppressing miR-27a expression

p53 is a major tumor suppressor whose function is pivotal for protection against cancer. In over half of human cancers, p53 is inactivated due to either point mutation or loss of p53 gene. It has been well established that in addition to abrogating the tumor-suppressive function of wild-type p53, mutant p53 gains new functions and actively contributes to various stages of tumor progression. However, little is known about whether microRNA (miRNA) is involved in the gain-of-function of mutant p53. Here we report miR-27a as a novel downstream transcriptional target of mutant p53-273H. Mutant p53 binds to the miR-27a promoter region and suppresses its expression. We also identify epidermal growth factor receptor (EGFR) as a direct target of miR-27a. Via the miR-27a/EGFR axis, mutant p53-273H promotes a sustained EGF-induced extracellular signal–regulated kinase 1/2 activation, thereby facilitating cell proliferation and tumorigenesis. Collectively, this work reveals a direct link between the gain-of-function of mutant p53 and miRNA and uncovers a novel mutant p53-273H/miR-27a/EGFR pathway that has an important role in promoting tumor development.

Sequential Administration of Bortezomib, Liposomal Doxorubicin and Dexamethasone (BDD) Followed by Thalidomide and Dexamethasone (TD) Results in Rapid Control of Untreated High-Risk Multiple Myeloma (MM) and Improves Depth of Response

Background: Doxorubicin and dexamethasone followed by thalidomide and dexamethasone is an effective, well-tolerated regimen for pts with MM. We previously reported an overall response rate (ORR) of 90.5%, with 35% achieving nCR/CR in 45 newly diagnosed pts, the majority of whom were ISS I (62%). Despite similar ORR, a survival analysis demonstrated inferior outcomes for pts with ISS II, ISS III or softtissue involvement by disease. Based on our experience, we designed a phase II study incorporating bortezomib and liposomal doxorubicin into the upfront treatment of pts with high-risk MM (ISS II, III or ISS I with soft-tissue disease). The aims of the study were to determine the safety and efficacy of bortezomib, pegylated doxorubicin and dexamethasone followed by thalidomide and dexamethasone (with or without bortezomib) in pts with untreated poor-risk or primary refractory MM. We correlated disease response with PET response to determine if there was concordance (or discordance) in these pts.Methods: 34 pts with high-risk MM have been enrolled. Treatment includes BDD (bortezomib 1.3mg/m2 IV days 1, 4, 8, and 11, liposomal doxorubicin 30mg/m2 IV on day 4, and dexamethasone 20mg PO on the day of and the day after bortezomib) for three 21-day cycles followed by 2 cycles of TD (thalidomide 200mg PO daily and dexamethasone 40mg PO on days 1–4, 9–12, 17–20) every 28 days for patients achieving at least PR, or BTD (less than a PR after BDD). Prophylactic use of acyclovir, fluconazole, and pyridoxine was implemented throughout the study. Aspirin 81mg or low molecular weight heparin (in patients with increased risk for VTE) was mandated during the TD portion of the study. Response was assessed using International Myeloma Working Group (IMWG) uniform response criteria. PET scans were performed at baseline, following BDD and at end of study (EOS) to determine the role of functional imaging in pts with high-risk MM.Results: At the time of this analysis, 28 pts have completed study and are evaluable. Baseline characteristics include median age of 58 years (range 41–80), 61% male, 43% (12/28) ISS II, 46% (13/28) ISS III, and 11% (3/28) ISS I with soft-tissue disease. Sixty-four percent of pts have abnormal cytogenetics: 39% (11/28) del 13q, 11% (3/28) t(4;14) and 7% (2/28) loss of p53 gene. Following BDD (1–3 cycles) the ORR was 79% with 25% CR/nCR and 36% ≥VGPR. At the EOS, the ORR remained 79%, however there is a significant improvement in the quality of response with 43% of pts achieving CR/nCR (p = 0.03) and 61% ≥VGPR (p = 0.008). Only 1 pt progressed on study based on skeletal survey findings despite achieving a serologic VGPR and improving clinically. Poor-risk cytogenetics (del 13q, t (4;14) or loss of p53) or soft-tissue disease did not affect response (p = 0.20 and p = 1.0, respectively). Of 11 pts with del 13q, 82% had ≥VGPR. Of the 5 pts with t (4;14) or loss of p53, 100% achieved ≥VGPR. There was poor agreement between EOS and PET response in the 19 pts who had PET data available, Kappa 0.11. The majority of pts (15/19) had PR by PET and 3 pts had SD. In the only pt that had progression by PET, IMWG response was VGPR and biopsy confirmed granulomatous disease rather than MM. Grades 3/4 neutropenia and thrombocytopenia occurred in 18% and 36% of pts, respectively. The incidence of neuropathy was 28%, but was grade 3 in only 1 pt (4%). Five pts developed DVT. Two pts developed asymptomatic decline in LVEF that improved with medical therapy. Fifty-seven percent of pts (16/28) have had successful stem cell harvest and have undergone ASCT. Three more pts have ASCT planned.Conclusions: BDD followed by TD (or BTD) results in rapid disease control with manageable toxicity in a poor-risk group of pts with MM. The depth of response is significantly improved with the sequential administration of novel agents, as has been demonstrated by other investigators (Jagannath ASH 2007). The 79% ORR is modest, however the ≥61% VGPR and 43% nCR/CR rates seen in these very high risk pts are comparable to initial therapy with VTD and Rev/Vel/Dex (Rajkumar ASH 2007 and Richardson ASH 2007, respectively) and are encouraging. High-quality responses were seen independent of cytogenetic abnormalities or the presence of soft-tissue disease. BDD followed by TD represents a promising therapeutic option for these pts. PET response does not correlate with IMWG response, perhaps due to the high sensitivity and lack of specificity of functional imaging.

Specific 5′CpG Island Methylation Signatures of FHIT and p16 Genes and Their Potential Diagnostic Relevance in Indian Breast Cancer Patients

Even after tremendous molecular studies, early detection,more accurate and sensitive diagnosis, and prognosis of breast cancer appear to be a riddle so far. To stab the enigma, this study is designed to envisage DNA methylation signatures as cancer-specific and stage-specific biomarkers in Indian patients. Rigorous review of scattered scientific reports on aberrant DNA methylation helped us to select and analyze a potential tumor suppressor gene pair (FHIT and p16 genes) in breast cancer patients. Methylation signatures from 232 primary sporadic breast cancer patients were pinpointed by methylation-specific PCR (MSP). To increase the sensitivity, we combined both MSP and expression studies (RT-PCR and Northern blotting) in a reproducible manner. Statistical analysis illustrated that hypermethylation of FHIT gene ( p < 0.0001) and p16 gene ( p=0.04) may be used as a potential diagnostic marker to diagnose the early and locally advanced stages of breast cancer. Additionally, the study authenticates the dependency of methylation and expressional loss of p16 gene on FHIT gene silencing. This observation not only describes the severity of disease when both genes are silenced but also drives to speculate the molecular cross talk between two genes or genetic pathways dictated by them separately.

Loss of p53 or p73 in human papillomavirus type 38 E6 and E7 transgenic mice partially restores the UV-activated cell cycle checkpoints

We have previously shown that human keratinocytes expressing E6 and E7 from the cutaneous human papillomavirus (HPV) type 38 have high levels of a specific form of p53, which in turn activate the transcription of DeltaNp73 gene. Expression of HPV38 E6 and E7 in mouse skin also promotes p53 and DeltaNp73 accumulation. Interestingly, keratinocytes of these mice do not undergo cell cycle arrest after skin ultraviolet (UV) irradiation. Here, we provide several lines of evidence that DeltaNp73 expression and lack of the UV response are directly linked. Loss of p53 gene in HPV38 E6/E7 transgenic mice abolished DeltaNp73 expression and partially restored the UV-activated cell cycle checkpoints. Similarly, loss of p73, and consequently DeltaNp73, led to restoration of the p53 pathways. In fact, keratinocytes of p73-/- HPV38 E6/E7 transgenic mice upon UV irradiation express high levels of p21(WAF1) and are cell cycle arrested. Thus, HPV38 E6 and E7, via DeltaNp73 accumulation, are able to alter the regulation of cell cycle checkpoints activated by UV radiation. These data suggest that UV and HPV may cooperate in skin carcinogenesis.

17p Deletion is associated with resistance of B-cell chronic lymphocytic leukemia cells to in vitro fludarabine-induced apoptosis

We explored the relationship between the cytogenetic/biologic characteristics of B-chronic lymphocytic leukemia (B-CLL) cells and their tendency to undergo spontaneous or fludarabine-induced apoptosis in vitro. B cells from 36 B-CLL patients were incubated with or without fludarabine for 48 h. Apoptosis was determined by two assays: annexin V staining and DNA staining. Fluorescence in situ hybridization was used for detection of trisomy 12, 11q deletion, and 17p deletion. Bcl-2 and CD38 expressions were determined by flow cytometry. Five patients had 17p deletion, 6 had trisomy 12, and another 6 had 11q deletion. B-CLL cells with 17p deletion had significant resistance to apoptosis induced by fludarabine and a slight spontaneous resistance to apoptosis. Bcl-2 and CD38 were not associated with in vitro spontaneous and fludarabine-induced apoptosis. In conclusion, 17p deletion, which causes loss of p53 gene, is associated with resistance to fludarabine-induced apoptosis in vitro. New treatment modalities should be tried in B-CLL patients with 17p deletion.

A high degree of aneuploidy, loss of p53 gene, and low soluble p53 protein serum levels are detected in ulcerative colitis patients.

The causes for the increased risk of colorectal cancer associated with ulcerative colitis have not been fully defined. Colonic tissue of ulcerative colitis patients was examined for changes in chromosome-17-centromere copy number, loss of the p53 gene, and alterations in serum levels of the 53-kDa protein. This study was performed under the assumption that these molecular events correlate with ulcerative colitis status and duration. Ulcerative colitis patients (n = 42) and healthy controls (n = 37) participated in the study. All participants were histopathologically and medically diagnosed. The stage of ulcerative colitis patients was stratified according to increasing risk factors for the development of colorectal cancer: left-sided colitis, pancolitis, sclerosing cholangitis, and dysplasia-associated lesions or masses. Changes in centromere number of chromosome 17 alone or in association with changes in copy number of the p53 gene were analyzed in colon tissue biopsies by fluorescence in situ hybridization. Serum p53 level was determined in blood samples by immunoprecipitation followed by separation using high-pressure liquid chromatography. Changes in chromosome 17 and p53 copy number and lower levels of serum p53 protein in ulcerative colitis patients directly correlated with colorectal cancer risk factors. All values significantly differed from controls. Significant direct correlations were obtained for ulcerative colitis disease duration, levels of p53 in the serum, and extent of aneuploidy. We demonstrate that in the colonic mucosa of ulcerative colitis patients, high levels of genomic instability, changes in p53 gene copy number, and lower levels of p53 in the serum directly correlate with the extent of disease duration and increased risk factors for colorectal cancer. Any of the measurements described herein can provide an acceptable prognostic tool in the assessment of colorectal cancer risk in ulcerative colitis patients.

Evaluation of relationship between chromosome 22 and p53 gene alterations and the subtype of meningiomas by the interphase-FISH technique.

In this study, we investigated the relationship between genetic alterations such as chromosome 22 aneuploidy and p53 gene deletion, and the pathological types of meningioma of typical and aggressive forms. Thirty-four meningiomas (23 typical and 11 aggressive) were examined by application of fluorescence in situ hybridization (FISH) with chromosome 22 specific alpha satellite probe and a combination of p53 locus specific and chromosome 17 centromere specific alpha satellite probes, to evaluate the chromosome 22 aneuploidy and gain or loss of p53 gene along with chromosome 17. The results showed that, although chromosome 22 aneuploidy was seen in 7 out of 23 typical (30.4%) and 4 out of 11 aggressive meningiomas (36.3%), no p53 deletion was detected in typical meningiomas, and p53 deletion was detected in 3 out of 11 aggressive meningiomas (1 atypical and 2 malignant), which had recurrence. There were no simultaneous occurrences of p53 gene deletions between typical and aggressive meningiomas. The present findings indicate that the loss of chromosome 22 may be involved with tumorogenesis of typical and aggressive meningiomas, while p53 gene deletions may be involved with malignant progression and recurrence in the aggressive meningiomas.

Heavy-ion-induced mutations in the gpt delta transgenic mouse: effect of p53 gene knockout.

The influence of the loss of p53 gene on heavy-ion-induced mutations was examined by constructing a new line of transgenic mice, p53 knockout (p53(-/-)) gpt delta. In this mouse model, deletions in lambda DNA integrated into the mouse genome are preferentially selected as Spi(-) phages, which can then be subjected to molecular analysis. Mice were exposed to 10 Gy of whole-body carbon-ion irradiation. The carbon ions were accelerated to 135 MeV/u by the RIKEN Ring Cyclotron. The p53 defect markedly enhanced the Spi(-) mutant frequency (MF) in the kidneys of mice exposed to C-ion irradiation: the Spi(-) MF increased 4.4- and 2.8-fold over the background level after irradiation in p53(-/-) and p53(+/+) mice, respectively. There was no significant difference in the background Spi(-) MF between p53(-/-) and p53(+/+) mice. Sequence analysis of the Spi(-) mutants indicated that the enhancement of kidney Spi(-) MF in p53(-/-) mice was primarily due to an increase in complex or rearranged-type deletions. In contrast to the kidney, the p53 defect had no effect on the Spi(-) MF in liver: Spi(-) MF increased 3.0- and 2.7-fold after the irradiation in p53(-/-) and p53(+/+) mice, respectively. Our results suggest that p53 suppresses deletion mutations induced by heavy-ion irradiation in an organ-specific manner.

Estimating loss of the wild-type p53 gene by in situ hybridization of fine-needle aspirates from breast carcinomas.

TP53 mutations have been found in 16-64% of breast carcinomas. The aim of our study was to investigate loss of the wild-type TP53 gene by in situ hybridization (ISH) of fine-needle aspirates (FNAC) from breast carcinomas. The material consisted of FNAC from 33 breast carcinomas, with histologic specimens from 19 of the cases. Routine diagnostic smears were used for cytologic grading. ISH of the wild-type TP53 gene and chromosome 17 was performed on air-dried smears. Hybridization signals were counted in at least 100 nuclei, and the percentage for each signal number was calculated. FNAC from four fibroadenomas as well as cell preparations from five lymphocyte cultures were used as normal/benign controls. Cutoff for defining loss of p53 gene signals was set at 20% of cells with zero and one gene signal only. Concomitant p53 protein expression was determined on 20 histologic sections and eight additionally available air-dried smears. Loss of wild-type p53 gene was found in 20 carcinomas (60.6%). The rate of signal loss varied from 0.4% to 75.3% of the cells. All tumors with aneusomy of chromosome 17 revealed loss of p53 gene signals, as did 42% of the disome cases. Loss of wild-type p53 gene was present in 10 of 16 grade 1 cancers (62.5%), eight of 13 grade 2 tumors (61.5%), and two of four grade 3 cases. Signal loss did not correlate with p53 protein expression. In conclusion, subpopulations with loss of the wild-type p53 gene are a common finding in breast carcinomas; they are detected in more than 60% of the tumors, including grade 1 cancers.

Roles of the functional loss of p53 and other genes in astrocytoma tumorigenesis and progression

Loss of function of the p53 tumor suppressor gene due to mutation occurs early in astrocytoma tumorigenesis in about 30-40% of cases. This is believed to confer a growth advantage to the cells, allowing them to clonally expand due to loss of the p53-controlled G1 checkpoint and apoptosis. Genetic instability due to the impaired ability of p53 to mediate DNA damage repair further facilitates the acquisition of new genetic abnormalities, leading to malignant progression of an astrocytoma into anaplastic astrocytoma. This is reflected by a high rate of p53 mutation (60-70%) in anaplastic astrocytomas. The cell cycle control gets further compromised in astrocytoma by alterations in one of the G1/S transition control genes, either loss of the p16/CDKN2 or RB genes or amplification of the cyclin D gene. The final progression process leading to glioblastoma multiforme seems to need additional genetic abnormalities in the long arm of chromosome 10; one of which is deletion and/or functional loss of the PTEN/MMAC1 gene. Glioblastomas also occur as primary (de novo) lesions in patients of older age, without p53 gene loss but with amplification of the epidermal growth factor receptor (EGFR) genes. In contrast to the secondary glioblastomas that evolve from astrocytoma cells with p53 mutations in younger patients, primary glioblastomas seem to be resistant to radiation therapy and thus show a poorer prognosis. The evaluation and design of therapeutic modalities aimed at preventing malignant progression of astrocytomas and glioblastomas should now be based on stratifying patients with astrocytic tumors according to their genetic diagnosis.

Radiogenic transformation of human mammary epithelial cells in vitro.

Cancer induction by space radiations is a major concern for manned space exploration. Accurate assessment of radiation risk at low doses requires basic understanding of mechanism(s) of radiation carcinogenesis. For determining the oncogenic effects of ionizing radiation in human epithelial cells, we transformed a mammary epithelial cell line (185B5), which was immortalized by benzo(a)pyrene, with energetic heavy ions and obtained several transformed clones. These transformed cells showed growth properties on Matrigel similar to human mammary tumor cells. To better understand the mechanisms of radiogenic transformation of human cells, we systematically examined the alterations in chromosomes and cancer genes. Among 16 autosomes examined for translocations, by using fluorescence in situ hybridization (FISH) technique, chromosomes 3, 12, 13, 15, 16, and 18 appeared to be normal in transformed cells. Chromosomes 1, 4, 6, 8, and 17 in transformed cells, however, showed patterns different from those in nontransformed cells. Southern blot analyses indicated no detectable alterations in myc, ras, Rb, or p53 genes. Further studies of chromosome 17 by using in situ hybridization with unique sequence p53 gene probe and a centromere probe showed no loss of p53 gene in transformed cells. Experimental results from cell fusion studies indicated that the transforming gene(s) is recessive. The role of genomic instability and tumor suppressor gene(s) in radiogenic transformation of human breast cells remains to be identified.

Highly Frequent Homozygous Deletion of the p16 Gene in Esophageal Cancer Cell Lines

To study the involvement of the p16 tumor suppressor gene in esophageal cancer development, we examined homozygous deletion of the p16 gene in 13 human esophageal cancer cell lines and 9 gastric cancer cell lines, in which some of genetic alterations have already been characterized. By Southern blot analysis, homozygous deletion was observed in 12 out of the 13 esophageal cancer cell lines (92%), in 2 out of a total of 9 gastric cancer cell lines (22%). It was also found that the p16 gene loss, cyclin D1 amplification and p53 gene mutations occurred independently in these cell lines. These findings suggest that loss or mutations of the p16 gene are involved in most esophageal cancers and that mutation of this gene plays a critical role in the development of esophageal cancer.

Infrequent loss of heterozygosity and mutation of the p53 gene in immortal and transformed mouse embryo fibroblasts.

Some of the progeny of isolated mouse embryo fibroblasts acquire the ability to grow indefinitely during cultivation, presumably through some mutational events. The relevance of p53 mutations and loss of heterozygosity to the mechanism of such immortal growth capability remains controversial. Since four bases in intron 1 of the p53 gene in C3H/HeJ mice are replaced by 13 different bases in DBA/2J mice, it is possible to distinguish maternal and paternal p53 alleles in the cells of F1 hybrids of these strains (C3D2F1) by electrophoresis of polymerase chain reaction fragments including the region. We established 23 spontaneously immortalized fibroblast cell lines from C3D2F1 mouse embryos and 29 transformed cell lines induced from one of the immortal cell lines, either by treatment with chemical carcinogens or by transfection with the c-Ha-ras gene. Of these 52 cell lines, only one, derived from fibroblasts unpassaged for 4 mo, showed p53 gene loss of heterozygosity and a structural alteration in the remaining allele. Our results demonstrated that p53 mutations are not a strict requirement for immortalization and transformation of mouse embryo fibroblasts in vitro.

P53 protein overexpression and allele loss of p53 gene in gastric adenocarcinoma.

Gene alterations of p53 tumor suppressor gene such as point mutations, deletions or insertions occur in various human cancers. p53 protein overexpression was studied immunohistochemically in 80 gastric adenocarcinomas using an anti-human p53 antibody (Pab 1801) and the avidin-biotin-peroxidase technique. We have also analyzed allele loss of the human p53 gene in 54 cases of gastric adenocarcinoma using polymerase chain reaction and restriction fragment length polymorphism. p53 immunostaining was also demonstrated in 48 of 80 carcinomas (60%). Normal mucosa was always negative. No relation could be found between p53 immunostaining and the degree of differentiation. 21 of the 54 patients(39%) were informative for the p53 exon 4. In ten of these informative cases(47.6%), tumor DNAs showed allele loss when compared with nonmetastatic lymph node DNAs. Seven of the ten(70%) showed p53 immunoreactivity. These findings suggest that mutations of the p53 gene may play a role in the development of gastric adenocarcinoma and that allele loss of p53 frequently occurs in p53 immunoreactive gastric adenocarcinoma.

Total loss of p53 DNA sequences in acute myeloid leukaemia

Mutations of the p53 tumour suppressor gene on chromosome 17p are a common genetic change in the malignant progression of many cancers. Here we report a case of a 71-year-old man with haematological, cytofluorimetric and cytochemical findings consistent with a ‘ de novo’ M2 acute myeloid leukaemia (AML). A complex karyotype including a whole chromosome 17 and a t(17;?) (p11;?) was present in 8 of 10 metaphases of bone marrow cells. Southern blot analysis of the bone marrow DNA showed a specific loss of p53 gene in the AML cells. As far as we know, this is the first report of a deletion of both p53 alleles in leukaemia. The effect of the loss of p53 on the course of AML is discussed.

Tumor Suppressor Gene Allelic Loss in Human Renal Cancers

It is now apparent that multiple genetic alterations, including oncogene activation and tumor suppressor gene inactivation, are necessary steps in carcinogenesis. We have studied this concept in renal cancers by looking at specific tumor suppressor genes implicated in several allelotyping studies. Primary, predominantly low stage renal tumors of varying grades and histologic subtypes were investigated for allelic loss of 3p, 17p and the p53 gene, the DCC gene and the Rb gene and its product. 3p loss occurred in 47% of tumors studied and was much more common in clear cell cancers (85%). 17p and p53 gene loss were relatively uncommon events with only 6 of 42 tumors demonstrating loss. None of the tumors with typical histologies had allelic loss of the DCC gene, though loss did occur in leiomyosarcoma and a collecting duct tumor. Allelic loss of the Rb gene occurred in one clear cell tumor, the leiomyosarcoma, and, interestingly, in both collecting duct tumors in this series. Allelic loss of the Rb gene was correlated with little or no RB protein expression as judged by immunohistochemistry. At all loci studied, allelic loss did not appear to correlate with tumor grade or stage. These results suggest that inactivation of the p53, Rb, and DCC genes by allelic loss are uncommon events in the early stages of renal carcinogenesis.