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F H Sarkar

Publications and source records attributed to F H Sarkar.

At least 73 records · Page 4Linked to original sources
PubMed1997

p53 status and prognosis of locally advanced prostatic adenocarcinoma: a study based on RTOG 8610.

BACKGROUND: The p53 tumor suppressor gene (also known as TP53) is one of the most frequently mutated genes in human cancer. Several studies have shown that p53 mutations are infrequent in prostate cancer and are associated with advanced disease. PURPOSE: We assessed the prognostic value of identifying abnormal p53 protein expression in the tumors of patients with locally advanced prostate cancer who were treated with either external-beam radiation therapy alone or total androgen blockade before and during the radiation therapy. METHODS: The study population consisted of a subset of patients entered in Radiation Therapy Oncology Group protocol 8610 ("a phase III trial of Zoladex and flutamide used as cytoreductive agents in locally advanced carcinoma of the prostate treated with definitive radiotherapy"). Immunohistochemical detection of abnormal p53 protein in pretreatment specimens (i.e., needle biopsies or transurethral resections) was achieved by use of the monoclonal anti-p53 antibody DO7; specimens in which 20% or more of the tumor cell nuclei showed positive immunoreactivity were considered to have abnormal p53 protein expression. Associations between p53 protein expression status and the time to local progression, the incidence of distant metastases, progression-free survival, and overall survival were evaluated in univariate (logrank test) and multivariate (Cox proportional hazards model) analyses. Reported P values are two-sided. RESULTS: One hundred twenty-nine (27%) of the 471 patients entered in the trial had sufficient tumor material for analysis. Abnormal p53 protein expression was detected in the tumors of 23 (18%) of these 129 patients. Statistically significant associations were found between the presence of abnormal p53 protein expression and increased incidence of distant metastases (P = .04), decreased progression-free survival (P = .03), and decreased overall survival (P = .02); no association was found between abnormal p53 protein expression and the time to local progression (P = .58). These results were independent of the Gleason score and clinical stage. A significant treatment interaction was detected with respect to the development of distant metastases: Among patients receiving both radiation therapy and hormone therapy, those with tumors exhibiting abnormal p53 protein expression experienced a reduced time to the development of distant metastases (P = .001); for patients treated with radiation therapy alone, the time to distant metastases was unrelated to p53 protein expression status (P = .91). CONCLUSIONS: Determination of p53 protein expression status yield significant, independent prognostic information concerning the development of distant metastases, progression-free survival, and overall survival for patients with locally advanced prostate cancer who are treated primarily with radiation therapy. IMPLICATIONS: The interaction of radiation therapy plus hormone therapy and abnormal p53 protein expression may provide a clinical link to experimental evidence that radiation therapy and/or hormone therapy act, at least in part, by the induction of apoptosis (a cell death program) and suggests that this mechanism may be blocked in patients whose tumors have p53 mutations.

Adenocarcinoma↗
PubMed1997

Relationship of family cancer history to the expression of p53, p21WAF-1, HER-2/neu, and K-ras mutation in pancreatic adenocarcinoma.

CONCLUSION: In our series of 81 cases, a history of family cancer was present in 52% of patients (42/81) with pancreatic cancer. Nine percent (7/81)had a family history of pancreatic cancer. Our studies suggest a possible relationship of family cancer history to the expression of p53 and p21WAF in pancreatic tumors, but show no relationship to the expression of HER-2/neu or to the prevalence of K-ras mutations. A lower incidence of p53 expression observed in patients with a family history of cancer suggests normal p53 protein is present in a majority of patients who develop pancreatic tumors related to other--as yet unidentified-inherited or familial risk factors. There was no significant difference in survival of pancreas cancer patients with and without a family history of cancer. However, survival in pancreas cancer patients may be influenced (improved) by p21WAF-1 expression. BACKGROUND: Pancreas cancer is the fifth leading cause of cancer deaths (27,800 deaths/yr) in the United States. Various risk factors, including cigarette smoking, high-fat diet, DDT exposure, chronic pancreatitis, and diabetes mellitus, have been associated with pancreatic carcinoma. A few studies have suggested a genetic predisposition or increased risk for pancreatic cancer within families, but the exact etiology is largely unknown. In a series of 81 patients with pancreatic carcinoma, we analyzed the status of K-ras gene mutations and the expression of P21WAF-1, p53, and HER-2/neu protein to identify possible molecular associations in pancreas cancer cases of these molecular markers to family histories of cancer and pancreas cancer. METHODS: Paraffin-embedded tissue sections from 81 cases of pancreatic adenocarcinoma were used for DNA extraction and immunohistochemical staining. K-ras mutation was studied by single-stranded conformation polymorphism (SSCP) and slot-blot allele-specific oligonucleotide (ASO) hybridization of PCR-amplified DNA product. Overexpression (aberrant expression) of p53, p21WAF-1, and HER-2/neu was documented by scoring nuclear localized p53, p21WAF-1 protein and cell membrane expression of HER-2/neu after immunostaining with gene product-specific monoclonal antibodies (MAbs). RESULTS: Forty-two (42) of 81 patients studied in this series had a history of cancer in their families (52%). Seven of those 42 had a history of pancreatic carcinoma (17% or 9% of total cases). The incidence of K-ras mutation and the expression of p21WAF-1 and HER-2/neu in patient groups with and without a family history of cancer was not statistically different (83 vs 74%, p = 0.416; 57 vs 41%, p = 0.184; and 83 vs 81%, p = 1.000, respectively). However, the incidence of p53 expression was significantly lower in patients with a family history of cancer (40 vs 72%, p = 0.007). There was no statistical difference in survival of patients with a family history of cancer in relation to either K-ras mutation, p53 expression, p21, or HER-2/neu expression. However, patients lacking a family history of cancer showed improved survival trends in relation to p21 expression (median survival of 16 vs 8 mo, p = 0.029).

Adenocarcinoma↗
PubMed1997

Prevalence and clinical significance of combined K-ras mutation and p53 aberration in pancreatic adenocarcinoma.

CONCLUSION: This study could not attribute survival differences to the coincident acquisition of two common genetic alterations, K-ras mutation and p53 overexpression in pancreatic adenocarcinoma patients. Additionally, our data indicate the converse to be true: Those patients lacking both K-ras mutation and aberrant p53 expression showed the shortest survival when compared with cases showing either alteration or both. This study also showed the negative effect of K-ras mutation and p53 expression on pancreas cancer patients' survival after treatment with either radiation therapy or chemotherapy. BACKGROUND: Mutations of the oncogene K-ras at codon 12 are reported to be the most common genetic alteration in pancreatic carcinoma, whereas either overexpression or mutation of the tumor suppressor p53 gene is considered the most common genetic alteration in neoplasia of all types. p53 overexpression has been attributed to survival differences in pancreatic carcinoma, but such association is still controversial. No studies have fully documented the combined incidence of K-ras and p53 alterations in pancreatic adenocarcinoma, or their combined effect on patient survival in a large case series. The influence of radiation or chemotherapy in groups showing both, either, or neither mutation is also undocumented. METHODS: Paraffin-embedded tissue sections from 76 cases of pancreatic adenocarcinoma were cut for DNA extraction for K-ras analysis and immunohistochemical staining for aberrant p53 expression. K-ras mutation was determined by single-strand conformation polymorphism (SSCP) and slot-blot allele-specific oligonucleotide (ASO) hybridization of PCR-amplified DNA product p53 expression was scored on the basis of percent nuclear staining with the MAb DO7. RESULTS: Sixty-four of 76 cases (84%) showed K-ras mutation, p53 expression, or both, K-ras was mutated in 55 of 76 cases (72%). p53 was expressed in 33 of 76 cases (43%). Twenty-four of 76 cases (31%) showed both K-ras mutation and p53 expression. The presence of both alterations was not related to significant differences in tumor grade, stage, or survival compared to either alteration alone. A sizable subset (16% of cases) lacked either alteration, and surprisingly, this group showed the shortest median survival compared to those with K-ras mutation, p53 expression, or both (p = 0.024). Patients whose tumors were K-ras-negative showed the greatest difference in median survival with radiation therapy (median survival 30.8 mo vs 7.8 mo with no radiation, p = 0.005).

Adenocarcinoma↗
PubMed1997

Clonal analysis of gliomas.

In malignant gliomas, the characteristically heterogeneous features and frequent diffuse spread within the brain have raised the question of whether malignant gliomas arise monoclonally from a single precursor cell or polyclonally from multiple transformed cells forming confluent clones. Although monoclonality has been shown in surgically resected tissues, these may not include the full spectrum of patterns seen on autopsy material. Little is known about the clonality of low-grade gliomas from which malignant gliomas may sometimes arise. We sought to investigate the clonality of low-grade and malignant gliomas by using and comparing surgical and autopsy material with a Polymerase chain reaction (PCR)-based assay for nonrandom X chromosome inactivation. For that, purpose, archival surgical and autopsy material from 15 female patients (group A) (age 4 to 73 years; median, 45) with malignant gliomas (12 glioblastomas, one gliosarcoma, one anaplastic oligoastrocytoma, one gliomatosis cerebri), surgical material only from 21 female patients (group S) (age 6 to 78 years; median, 60) with low-grade and malignant gliomas (four low-grade astrocytomas, three oligoastrocytomas, two anaplastic astrocytomas, one gemistocytic astrocytoma, four oligodendrogliomas, seven glioblastomas) were analyzed. In group A, representative areas (mean = 5/patient; median = 7) were microdissected from tissue sections and assayed by PCR amplification of a highly polymorphic microsatellite marker locus of the human androgen receptor gene (HUMARA) in the presence of alpha32P with and without predigestion with a methylation-sensitive restriction enzyme (HhaI). Products were resolved by denaturing gel electrophoresis and autoradiographed. In group S, selected tumor areas were used for the assay. Each patient's normal brain tissue was used for control. The band intensity of alleles were measured by densitometric scanning. In group A, 13 of 15 cases were informative (heterozygous). The same pattern of nonrandom X chromosome inactivation was present in all areas of solid dense and moderate tumor infiltration in eight including all components of the gliosarcoma. Two of eight also showed focal loss of heterozygosity (LOH). One of 13 presented global LOH. Two of 13 showed microsatellite instability, one of which in a patient with Turcot syndrome, the other in gliomatosis cerebri. Opposite skewing patterns were seen in distant areas of gliomatosis cerebri consistent with oligoclonal derivation. Clonality remained indeterminate in one glioblastoma and in the anaplastic oligoastrocytoma because of skewed lyonization in the normal control. In group S, 19 of 21 cases were informative. Fifteen of 19 were monoclonal (four low-grade astrocytomas, one anaplastic astrocytoma, one gemistocytic astrocytoma, two oligodendrogliomas, one oligoastrocytoma, six glioblastomas). Four of 19 were indeterminate. We conclude that (1) Low-grade and malignant gliomas are usually monoclonal tumors, and extensively infiltrating tumors must result from migration of tumor cells (2) Gliomatosis cerebri may initiate as an oligoclonal process or result from collision gliomas (3) Biphasic gliomas likely arise from a single precursor cell. (4) LOH at the HUMARA locus is probably related to partial or complete deletion of an X-chromosome, which occurs in malignant gliomas during clonal evolution.

Adolescent↗
PubMed1997

Clinicopathologic analysis of amphiregulin and heregulin immunostaining in breast neoplasia.

We observed no association between neoplastic epithelial immunostaining for either amphiregulin (AR) or heregulin (HRG) and presence of ER, EGFR/ERBB-2 overexpression, nodal status, or disease recurrence in 34 breast carcinomas. However, stromal cell staining for both correlated with outcome; 29% of stromal cell AR - cases recurred vs. 85% for AR + cases (p = 0.001), and 41% of stromal cell HRG - cases recurred vs. 82% of HRG + cases (p = 0.01). We conclude that both HRG and AR have significant biologic roles in breast carcinoma growth or progression via mediation of host-tumor interactions which favor aggressive tumor behavior.

Amphiregulin↗
PubMed1997

HER-2/neu expression in pancreatic adenocarcinoma: relation to tumor differentiation and survival.

HER-2/neu expression in pancreatic adenocarcinoma has been inconsistently reported and has not been fully evaluated with respect to histologic grade and tumor grade heterogeneity. We studied HER-2/neu expression in a series of 79 primary pancreatic carcinomas using immunohistochemical methods, with expression scored for each histologic grade represented in each tumor. We found significantly lower expression of HER-2/neu in poorly differentiated (PD) portions of tumors-those areas lacking glandular differentiation-compared to well-differentiated (WD) and moderately differentiated (MD) portions of tumors. Forty-two of 68 (62%) invasive tumors with WD or MD glands showed moderate or strong expression of HER-2/neu in WD/MD areas; only 6 of 32 (19%) invasive tumors with PD areas showed similar expression in PD. In mutually exclusive patient sets, we also found a statistically different prevalence of HER-2/neu expression in patients with PD (6/32 cases; 19%) and without PD (29/47 cases; 62%) tumors (p < 0.001). Twenty-three cases had directly comparable areas of PD versus MD or WD. In 19 of 23 cases HER-2/neu expression was graded comparatively lower (or negative) in areas of PD than in MD or WD. Overall 46 of 79 cases (58%) showed moderate to strong HER-2/neu expression inclusive of all histologic grades, and 63 of 79 (80%) cases were HER-2/neu positive, if including weak or focal staining. There was no significant difference in the survival of patients with HER-2/neu-positive versus-negative tumors or in patients with versus without PD tumors. We have confirmed that although HER-2/neu gene expression is common to many pancreatic carcinomas, it is not common to tumors lacking glandular differentiation. HER-2/neu gene expression could not be related to survival differences--perhaps due to overall poor survival within adenocarcinomas of the pancreas--but the pattern of HER-2/neu expression suggests a relationship to glandular differentiation and early oncogenesis.

Adenocarcinoma↗
PubMed1997

Clinicopathologic analysis of k-ras, p53, and ERBB-2 gene alterations in pulmonary adenocarcinoma.

We compared PCR-SSCP detected mutations of k-ras (codon 12) and p53 (exons 5-8) to ERBB-2 immunostaining and clinicopathologic features in 31 pulmonary adenocarcinomas. There were nine tumors (29%) with mutations of ras, 13 tumors (42%) with mutations of p53, and three tumors (10%) with mutations of both. Neither k-ras nor p53 mutation alone was significantly correlated with stage, grade, or survival. However, tumors with k-ras mutation were more frequently associated with an invasive growth pattern, defined as > 30% tumor volume composed of infiltrative nests of cells within desmoplastic, scar-like stroma [< 30% volume invasive--1/13 (8%) with k-ras mutation vs. > 30% volume invasive--8/18 (44%) with k-ras mutation, p = 0.02]. Accordingly, k-ras mutations were observed in only 1/9 (15%) predominantly bronchoalveolar or papillary tumors versus 6/22 (28%) acinar or scar carcinoma tumors. All three patients with combined k-ras/p53 mutation had advanced stage (III/IV) at presentation and died of the disease. In contrast to k-ras, staining for ERBB-2 was more frequently observed in tumors exhibiting < 30% invasive growth pattern (12/13, 92%) than in tumors with > 30% invasive growth pattern (10/18, 56%, p = 0.03). ERBB-2 immunoreactivity was more frequent in Stage I (14/15, 93%) versus Stage II-IV (8/16, 50%) cases, but it did not correlate with survival. There was a reciprocal relationship between k-ras mutation and ERBB-2 staining; only 4/9 (44%) k-ras mutated cases were ERBB-2 positive versus 18/22 (82%) cases without k-ras mutation (p = 0.005). In contrast, 8/13 cases with p53 mutation were ERBB-2 positive. We conclude that well-differentiated and less invasive papillary and bronchoalveolar tumors are more often ERBB-2 positive/k-ras negative (i.e. at codon 12), whereas less well differentiated acinar or scar carcinomas are more often ERBB-2 negative/k-ras mutated at codon 12. These findings imply that the divergent histogenesis of pulmonary adenocarcinoma may reflect specific differences in genetic pathology.

Adenocarcinoma↗
PubMed1997

Increased expression of EGFR in gastric mucosa of aged rats.

Although in Fischer-344 rats aging is found to be associated with increased gastric mucosal proliferative activity, little is known about the intracellular events that regulate this process. The present investigation examines the age-related changes in gastric mucosal tyrosine kinase activity and expression of epidermal growth factor receptor(EGFR) and its structural and functional analog p185c-erbB-2, the protein product of c-erbB-2/c-neu protooncogene. We observed a significantly higher intrinsic tyrosine kinase activity and tyrosine phosphorylation of EGFR and p185c-erbB-2 in the gastric mucosa of 24-mo-old (aged) rats than in that of their 4- or 12-mo-old counterparts. This was associated with increased levels of EGFR protein and steady-state mRNA levels of EGFR and p185c-erbB-2. In addition, we also observed threefold higher steady-state mRNA levels of transforming growth factor-alpha (TGF-alpha; one of the primary ligands of EGFR) in the gastric mucosa of aged rats than in that of 4-mo-old (young) animals. This was accompanied by a fivefold increase in the relative concentration of the 18-kDa precursor form of TGF-alpha in gastric mucosal membranes but not in the cytosol. In conclusion, our data demonstrate that aging is associated with increased tyrosine kinase activity of EGFR and p185c-erbB-2 in the gastric mucosa. Moreover, the observation that aging results in increased accumulation of TGF-alpha in gastric mucosal membranes raises the possibility that the membrane-bound TGF-alpha could partly be responsible for the constitutively active EGFR-induced signaling pathway in the gastric mucosa of aged rats and, in turn, for stimulation of mucosal proliferative activity.

Aging↗
PubMed1996

Differential growth factor responses of epithelial cell cultures derived from normal human prostate, benign prostatic hyperplasia, and primary prostate carcinoma.

Because of a lack of information of the optimum nutritional requirements, epithelial cells derived from normal human prostate and prostate tumors have been difficult to propagate in vitro, which hinders research in prostate carcinogenesis. In an effort to establish optimum nutritional conditions and differences in growth characteristics of normal human prostate (NP), benign prostatic hyperplasia (BPH), and prostatic carcinoma (PCA), we have compared the effects of several growth factors on cell proliferation and elucidated growth properties of low passage epithelial cells derived from NP, BPH, and PCA of an African-American patient. Primary and low passage cultures were propagated in serum-free keratinocyte basal medium (KBM) supplemented with insulin (5 micrograms/ml), hydrocortisone (0.5 microgram/ml), epidermal growth factor (EGF, 10 ng/ml), bovine pituitary extract (BPE; 50 micrograms/ml), cholera toxin (10 ng/ml), and antibiotics. Almost all NP, BPH, and PCA cells were positive for cytokeratins and prostate-specific antigen (PSA). The NP, BPH, and PCA cells were essentially diploid and lacked mutations in c-K-ras and c-Ha-ras oncogenes, and p53 tumor suppressor gene. However, they exhibited progressively accelerating growth parameters. The population doubling times of NP, BPH and PCA were 51 hr, 37 hr, and 29 hr, respectively; their saturation densities were 2.9 x 10(4)/cm2, 3.3 x 10(4)/cm2, and 7.2 x 10(4)/cm2, respectively. The NP and BPH cells required all of the growth factors in the medium, as deletion of any one of the above factors strongly inhibited their growth. The PCA cells, however, were independent of EGF and hydrocortisone. PC-3, an established human prostate cancer cell line, was independent of the growth factors tested. Fetal bovine serum (FBS) inhibited the growth of NP, BPH and PCA cells. In contrast, FBS stimulated the growth of the PC-3 cells in a concentration-dependent manner. These results indicate that in the absence of any apparent karyotype alterations and mutations in c-K-ras, c-Ha-ras and p53 genes, epithelial cells derived from NP, BPH, and PCA exhibit significant differences in their growth properties and responses to growth factors. These variations may represent early changes involved in prostate cancer, while gene mutations and cytogenetic alterations occur in advanced and/or metastatic tumors.

Black or African American↗
PubMed1996

Remodelling of adult cardiac muscle cells in culture: dynamic process of disorganization and reorganization of myofibrils.

The myofibrils of adult rat cardiac muscle cells in long-term culture initially break down and later reassemble into mature myofibrils. The objective of this study is to examine the disorganization process of myofibrils and to determine how disorganized myofibrillar proteins, myosin, titin, actin, and alpha-actinin are reorganized into mature myofibrils in adult cells. After dismantlement of myofibrils during initial culture period (24-72 h), myofibrillar proteins became disorganized into amorphous form. These proteins later were observed in vesicular, amorphous, and nonstriated fibrillar forms. Some vesicular structures, containing mainly myosin, titin, alpha-actin, and alpha-actinin were observed on the outer surfaces of the cell and outside the cell body. Such vesicles containing F-actin were rare. Punctate structures of alpha-actinin emerged from the pre-existing amorphous alpha-actinin along with the appearance of mostly titin periodicities. The periodicities of alpha-actinin later became prevalent, followed by the appearance of periodicities of actin. alpha-actinin provided an initiation point on which titin and actin became associated, forming titin-associated I-Z-I structures. Titin traversed the I-bands on either side of the Z-line. The phalloidin-stained I-Z-I structures bound to antibodies to muscle specific sarcomeric proteins (titin, alpha-actin, alpha-actinin). The differentiation of myosin periodicities lagged behind those of titin, alpha-actinin, and actin although presarcomeric structures of immunolabelled titin and myosin were very closely linked in their distributions in the formative myofibrils. Variations in the temporal sequence of emergence of periodicities of alpha-actinin and myosin were observed among certain myocytes. Also observed was the variation of the temporal sequence of emergence of titin and actin periodicities among different myocytes and within a single myocyte. Even in the late stage of culture (30 days), when the cell body was packed with myofibrils, the myocytes contained remnants of amorphous myofibrillar proteins.

Actinin↗
PubMed1996

Correlation between p53 immunostaining patterns and gene sequence mutations in breast carcinoma.

We performed p53 immunostaining in 82 invasive breast carcinomas by using two commercially available antibodies, one of which (DO7) was employed in formalin-fixed paraffin-embedded sections. The other antibody (PAb1801) was evaluated in corresponding acetone-fixed cryostat sections. A greater percent of cases were immunostained with DO7 compared to PAb1801 (52% vs 33%); however, the staining was more often heterogeneous (6-50% cells positive) or focal (< or = 5% cells positive) with DO7 (9% vs 31%). To investigate the genetic relevance of p53 immunostaining, single-strand conformational polymorphism (SSCP) analysis and DNA sequencing were performed on exons 2-11 by using archival tissue samples of 18 cases that were selected on the basis of certain immunostaining patterns. Two (33%) of six tumors with negative staining for DO7 had gene sequence mutations; however, one of these mutations was a base-pair deletion that caused a reading-frame shift and the other was a base-pair insertion that resulted in a stop codon. Both of these tumors exhibited immunostaining with PAb1801, although it was weak and cytoplasmic in one case. Conversely, three (30%) of 10 tumors showing immunoreactivity in 6-100% of cells with both reagents lacked a gene sequence mutation. Of the remaining seven tumors that were positive by SSCP, six contained a point mutation resulting in a base-pair substitution. Despite repeat analyses, one of the cases positive by SSCP failed to demonstrate a mutation in the sequenced exons. Four (80%) of five cases with heterogeneous DO7 immunoreactivity (that is, 6-50% of nuclei positive) were positive for gene sequence mutation. Neither of two cases showing focal DO7 nuclear staining in < 5% of tumor cells contained a mutation in the sequenced exons, and neither of these cases was strongly positive with PAb1801. Staining for either antibody was significantly associated with adverse outcome, as determined by disease recurrence at 52 months median follow-up (DO7, p = 0.01; and PAb1801 p = 0.002, chi-squared test). We conclude that a variety of factors may account for discrepancies when immunohistology is used to evaluate p53 status. These include fixation artifacts, differing epitope specificities of monoclonal reagents, presence of immunohistologically "silent" mutations and, possibly, aberrant overexpression of wild-type protein.

Antibodies, Monoclonal↗
PubMed1996

Tumor suppressor p53 gene mutation in squamous cell carcinoma of the larynx.

Using polymerase chain reaction (PCR)-SSCP (single-strand conformation polymorphism) and PCR-DNA sequencing analysis, we screened 61 cases of laryngeal squamous cell carcinoma for mutations in exons 5-8 of the p53 gene. Mutations were found in 31.3% (19 of 61) of the laryngeal cancers. Seventeen of 19 (84.2%) cases showing p53 gene mutations were stage III and IV, which suggests that p53 gene mutation is a rather late event in tumor development and is involved in the progression of laryngeal squamous cell carcinoma. A high frequency of G:C to T:A transversion (50%, seven of 14), especially G to T (35.7%, five of 14), was noted in laryngeal carcinoma samples in our study. This finding may point toward an environmental carcinogen (such as tobacco smoke) as an important agent in the genesis of laryngeal squamous cell carcinoma. Most of the p53 gene mutations (18 of 19) found in our studies could change the protein and thus may cause an inactivation of the p53 tumor suppressor gene, strongly suggesting that p53 gene mutation plays a crucial role in the progression of laryngeal carcinoma.

Biomarkers, Tumor↗
PubMed1995

bcl-2 suppresses expression of p21WAF1/CIP1 in breast epithelial cells.

The tumor suppressor gene p53 regulates G1 checkpoint prior to the initiation of DNA synthesis, which can either induce G1 arrest or signal apoptosis. The involvement of p53 in apoptosis may also be related to its ability to down-regulate transcription of the bcl-2 gene. The bcl-2 gene product prevents most types of apoptotic cell death, suggesting that bcl-2 interferes with an essential signaling molecule involved in the apoptotic cell death pathway. Although the bcl-2 protein is shown to be overexpressed in many types of human tumor including breast cancer, its biochemical or pathological consequences are poorly understood. To determine the effects of bcl-2 overexpression on apoptosis and transformation of breast epithelial cells and to investigate whether bcl-2 interferes with the p53 pathway, we introduced the bcl-2 expression vector into MCF10A cells, which were derived from diploid human breast epithelial cells containing the wild-type p53 gene. Overexpression of bcl-2 prevented free radical-induced apoptosis and induced a partially transformed phenotype in MCF10A cells. Although overexpression of bcl-2 did not affect the expression of the p53 gene, p53-dependent gene transcription such as p21WAF1/CIP1 was suppressed. These results suggest that bcl-2 may inhibit p53 functional activity and is involved in the regulation of an early commitment step either to proliferate or suicide.

Apoptosis↗
PubMed1995

BCR/ABL fusion located on chromosome 9 in chronic myeloid leukemia with a masked Ph chromosome.

A reciprocal translocation, t(10;22)(q22;q11), resulting in a masked Ph chromosome was identified in a patient diagnosed with chronic myeloid leukemia (CML). Both homologs of chromosome 9 were of the normal pattern. Two signals for the ABL probe, both of them hybridized to chromosome 9, were demonstrated via fluorescence in situ hybridization (FISH). Furthermore, cohybridization with two differently labeled BCR/ABL translocation DNA probes indicated a BCR/ABL fusion apparently located on 9q34. Molecular studies revealed a rearrangement of the BCR region and expression of a chimeric BCR/ABL mRNA of CML configuration. These findings indicate that the BCR/ABL fusion resulted from an unusual relocation of the BCR gene from its normal position on 22q11 to 9q34 adjacent to the ABL gene.

Chromosomes, Human, Pair 9↗
PubMed1995

Detection of M-bcr/abl fusion by fluorescence in situ hybridization (FISH) in a case of Ph negative CML.

A balanced reciprocal translocation, t(6;9)(p21;q34), was identified in a female patient diagnosed with chronic myeloid leukemia (CML). Both homologs of chromosome 22 were of normal length and morphology. Southern blotting revealed a bcr rearrangement with BglII and HindIII. Two signals for the abl probe were demonstrated by fluorescence in situ hybridization (FISH), one on the normal chromosome 9 and the second on a chromosome 22. Thus, molecular rearrangement of bcr resulted from insertion of an abl gene within the bcr region despite absence of a Ph chromosome.

Chromosomes, Human, Pair 6↗
PubMed1995

A universal method for the mutational analysis of K-ras and p53 gene in non-small-cell lung cancer using formalin-fixed paraffin-embedded tissue.

The p53 tumor suppressor gene has been found to be altered in almost all human solid tumors, whereas K-ras gene mutations have been observed in a limited number of human cancers (adenocarcinoma of colon, pancreas, and lung). Studies of mutational inactivation for both genes in the same patient's sample on non-small-cell lung cancer have been limited. In an effort to perform such an analysis, we developed and compared methods (for the mutational detection of p53 and K-ras gene) that represent a modified and universal protocol, in terms of DNA extraction, polymerase chain reaction (PCR) amplification, and nonradioisotopic PCR-single-strand conformation polymorphism (PCR-SSCP) analysis, which is readily applicable to either formalin-fixed, paraffin-embedded tissues or frozen tumor specimens. We applied this method to the evaluation of p53 (exons 5-8) and K-ras (codon 12 and 13) gene mutations in 55 cases of non-small-cell lung cancer. The mutational status in the p53 gene was evaluated by radioisotopic PCR-SSCP and compared with PCR-SSCP utilizing our standardized nonradioisotopic detection system using a single 6-microns tissue section. The mutational patterns observed by PCR-SSCP were subsequently confirmed by PCR-DNA sequencing. The mutational status in the K-ras gene was similarly evaluated by PCR-SSCP, and the specific mutation was confirmed by Southern slot-blot hybridization using 32P-labeled sequence-specific oligonucleotide probes for codons 12 and 13. Mutational changes in K-ras (codon 12) were found in 10 of 55 (18%) of non-small-cell lung cancers. Whereas adenocarcinoma showed K-ras mutation in 33% of the cases at codon 12, only one mutation was found at codon 13. As expected, squamous cell carcinoma samples (25 cases) did not show K-ras mutations. Mutations at exons 5-8 of the p53 gene were documented in 19 of 55 (34.5%) cases. Ten of the 19 mutations were single nucleotide point mutations, leading to amino acid substitution. Six showed insertional mutation, and three showed deletion mutations. Only three samples showed mutations of both K-ras and p53 genes. We conclude that although K-ras and p53 gene mutations are frequent in non-small-cell lung cancer, mutations of both genes in the same patient's samples are not common. We also conclude that this universal nonradioisotopic method is superior to other similar methods and is readily applicable to the rapid screening of large numbers of formalin-fixed, paraffin-embedded or frozen samples for the mutational analysis of multiple genes.

Base Sequence↗
PubMed1995

Antiintegrin alpha v beta 3 blocks human breast cancer growth and angiogenesis in human skin.

Angiogenesis plays a fundamental role in human breast tumor progression. In fact, recent findings indicate that vascular density is a prognostic indicator of breast cancer disease status. Evidence is presented that the integrin alpha v beta 3 is not only a marker of human breast tumor-associated blood vessels, but that it plays a significant role in human angiogenesis and breast tumor growth. To assess the role of alpha v beta 3-dependent angiogenesis in the progression of human breast cancer, we examined a SCID mouse/human chimeric model with transplanted full thickness human skin containing alpha v beta 3-negative human breast tumor cells. This tumor induced a human angiogenic response as measured by vascular cell immunoreactivity with monoclonal antibodies LM609 and P2B1 directed to human alpha v beta 3 and CD31, respectively. Intravenous administration of LM609 either prevented tumor growth or markedly reduced tumor cell proliferation within the microenvironment of the human skin. These LM609-treated tumors not only contained significantly fewer human blood vessels but also appeared considerably less invasive than tumors in control animals. These findings demonstrate that alpha v beta 3 antagonists may provide an effective antiangiogenic approach for the treatment of human breast cancer.

Animals↗