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J R Gnarra

Publications and source records attributed to J R Gnarra.

At least 37 records · Page 2Linked to original sources
PubMed1995

Loss of heterozygosity occurs centromeric to RB without associated abnormalities in the retinoblastoma gene in tumors from patients with metastatic renal cell carcinoma.

Tumor suppressor genes have been found to have loss of function in a number of malignancies. This loss of function is believed to contribute to malignant transformation or metastatic spread. In the present study, expression of the retinoblastoma (RB) tumor suppressor gene was examined in cell lines and tumor tissue obtained from primary renal and metastatic sites in patients with metastatic renal cell carcinoma. Three of fifteen (20%) of informative renal carcinoma cell lines had loss of heterozygosity (LOH) in the RB gene (intron 20) detected by polymerase chain reaction analysis. Using restriction fragment length polymorphism (RFLP) analysis, 7 of 22 (32%) informative cell lines had LOH centromeric to the RB gene at the D13S1 locus. No LOH (0 of 7) was seen telomeric to the RB gene at the D13S2 locus. None of the 28 cell lines examined had decreased RB mRNA expression compared with short-term cultures of proximal renal tubular cells. Western blotting demonstrated phosphorylated and unphosphorylated forms of RB protein of expected molecular weight in all 41 cell lines (33 primary and 8 metastatic) examined. Twenty-nine primary cell lines and 6 metastatic cell lines all demonstrated normal immunohistochemical staining. Loss of RB immunohistochemical staining in paraffin-embedded tissue was detected in none of the primary tumors (0 of 30) or metastatic tumors (0 of 12). The absence of abnormalities of RB expression detected in these renal cell carcinomas suggests that abnormalities of the RB gene are not central to malignant transformation or progression in this tumor type; however, another tumor suppressor gene centromeric to the RB locus may be important in renal cell carcinogenesis.

Base Sequence↗
PubMed1995

Renal cell carcinoma. Molecular genetics and clinical implications.

The combined efforts of a number of investigators have led to the identification of the VHL gene, which appears to function as a tumor suppressor gene and is implicated in both sporadic and familial forms of RCC. These findings should increase our understanding of the molecular biology of this malignancy; however, there is much work to be done. Identification of the mechanism of inactivation of the VHL gene, as well as the structure and function of the VHL gene product, ultimately may provide clinicians with greater understanding of this malignancy as well as with methods for earlier diagnosis. The role of other tumor suppressor genes, such as p53, is incompletely understood. It is hoped that the techniques that have been applied to the study of RCC also will result in advances in our knowledge of other urologic malignancies.

Aged↗
PubMed1995

Genetics of renal-cell carcinoma and evidence for a critical role for von Hippel-Lindau in renal tumorigenesis.

Using a genetic linkage-based positional cloning approach the VHL gene was identified at chromosome 3p25.5. VHL is mutated in the germlines of affected individuals, and in VHL-associated tumors the mutation is almost always exposed by virtue of chromosomal deletion of the inherited wild-type allele. VHL is also frequently mutated in sporadic, nonpapillary RCC and in familial RCC. This was predicted because such tumors are histologically similar to VHL-associated renal tumors. Knowledge that VHL plays a critical role in sporadic RCC should aid in the future diagnosis and treatment of this malignancy. Detailed analyses of the biology of individual mutations will be required to determine whether the inherited VHL mutations or acquired sporadic mutations cause loss of protein function or have dominant-negative affects. However, the nature of the VHL protein is at present unclear and a complete understanding its function will only be expected after the cloning of the full-length gene.

Carcinoma, Renal Cell↗
PubMed1994

Silencing of the VHL tumor-suppressor gene by DNA methylation in renal carcinoma.

Mutational inactivation and allelic loss of the von Hippel-Lindau (VHL) gene appear to be causal events for the majority of spontaneous clear-cell renal carcinomas. We now show that hypermethylation of a normally unmethylated CpG island in the 5' region provides another potentially important mechanism for inactivation of the VHL gene in a significant portion of these cancers. This hypermethylation was found in 5 of 26 (19%) tumors examined. Four of these had lost one copy of VHL while one retained two heavily methylated alleles. Four of the tumors with VHL hypermethylation had no detectable mutations, whereas one had a missense mutation in addition to hypermethylation of the single retained allele. As would be predicted for the consequence of methylation in this 5' CpG island, none of the 5 tumors expressed the VHL gene. In contrast, normal kidney and all tumors examined with inactivating VHL gene mutations but no CpG island methylation had expression. In a renal cell culture line, treatment with 5-aza-2'-deoxycytidine resulted in reexpression of the VHL gene. These findings suggest that aberrant methylation of CpG islands may participate in the tumor-suppressor gene inactivations which initiate or cause progression of common human cancers.

Adenocarcinoma, Clear Cell↗
PubMed1994

Loss of HLA haplotype and B locus down-regulation in melanoma cell lines.

The expression of HLA class I molecules on tumor cells is vital for CD8+T cell recognition of tumor Ags. Loss of HLA class I Ag expression as a result of defective beta 2-microglobulin genes has been described in melanoma cells. To further evaluate mechanisms of tumor escape, HLA class I Ag expression was compared in 24 metastatic melanoma cell lines and 20 melanocyte strains by FACS analysis with use of allele-specific mAbs. Total loss of HLA class I Ag expression was not noted; instead, two relatively common phenomena were identified: 1) A variable degree of expression of HLA-B Ags by melanoma cell lines and melanocytes; however, HLA-A Ags were consistently expressed in all cell types. Furthermore, HLA-B locus Ag expression was detected in vivo in only one of six frozen section specimens obtained from six patients having metastatic melanoma. 2) Loss of allelic expression was noted in two of 14 HLA-A2 (14%) and one of three HLA-A29 (33%) melanoma cell lines and included a full haplotype, which suggests loss of a genomic fragment. Allele-specific PCR amplification demonstrated deletion of genes in linkage disequilibrium within the MHC class II, III, and I regions. Aberrations of HLA class I expression in tumor lines should be considered when assessing MHC-restricted phenomena in in vitro models.

Alleles↗
PubMed1994

Loss of heterozygosity on the short arm of chromosome 3 in mesothelioma cell lines and solid tumors.

Cytogenetic analysis of mesothelioma cell lines and solid tumors has documented non-random chromosomal abnormalities on the short arm of chromosome 3 from 3p14 to 3p25. We therefore examined nine mesothelioma cell lines, their corresponding tumors, and 15 additional mesothelioma tumors for loss of heterozygosity on 3p from 3p13 to 3p25.5 by polymerase chain reaction and restriction fragment length polymorphism analysis at 8 loci: D3S3, D3S30, D3S6, D3S2, D3S32, D3F15S2, THRB, and VHL. Loss of heterozygosity was documented by loss of one of two alleles in the tumor DNA whose corresponding normal DNA was heterozygous and was documented in four of nine mesothelioma cell lines and six of 15 mesothelioma tumors or a total of 42% of the mesotheliomas evaluated. This study suggests the involvement of a gene on the short arm of chromosome 3 in the development of mesotheliomas.

Alleles↗
PubMed1994

The use of molecular genetic analysis in the diagnosis of renal cell carcinoma.

The most common genetic aberration seen in nonpapillary renal cell carcinoma is believed to be the loss or inactivation of allelic material on the short arm of chromosome 3 (3p). Two patients underwent nephrectomy at our institution, each initially receiving a histologic diagnosis of renal cell carcinoma. Molecular analysis of these tissues revealed no genetic deletion on 3p, prompting further histologic and immunohistochemical evaluation of the original specimens. These additional studies confirmed alternate histologies other than renal cell carcinoma in each case. These data suggest that the loss of genetic material on the short arm of chromosome 3 may be specific for nonpapillary renal cell carcinoma. Molecular analysis may prove to be a useful adjunct in establishing the diagnosis in renal neoplasms with equivocal histologies.

Adult↗
PubMed1994

Mutations of the VHL tumour suppressor gene in renal carcinoma.

Multiple, bilateral renal carcinomas are a frequent occurrence in von Hippel-Lindau (VHL) disease. To elucidate the aetiological role of the VHL gene in human kidney tumorigenesis, localized and advanced tumours from 110 patients with sporadic renal carcinoma were analysed for VHL mutations and loss of heterozygosity (LOH). VHL mutations were identified in 57% of clear cell renal carcinomas analysed and LOH was observed in 98% of those samples. Moreover, VHL was mutated and lost in a renal tumour from a patient with familial renal carcinoma carrying the constitutional translocation, t(3;8)(p14;q24). The identification of VHL mutations in a majority of localized and advanced sporadic renal carcinomas and in a second form of hereditary renal carcinoma indicates that the VHL gene plays a critical part in the origin of this malignancy.

Adenocarcinoma, Clear Cell↗
PubMed1994

Molecular analysis of the von Hippel-Lindau disease tumor suppressor gene in human lung cancer cell lines.

The deletion of the short arm of chromosome 3 is frequently observed in lung cancer. To determine whether the von Hippel-Lindau (VHL) disease tumor suppressor gene located at 3p25 is responsible for oncogenesis in lung cancer, we searched the known open reading frame using the single-strand conformation polymorphism (SSCP) technique for mutations in the VHL gene in 72 cancer cell lines including small cell (SCLC) and non-small cell (NSCLC) lung cancers, carcinoids, and mesotheliomas. SSCP analysis showed that four cell lines have altered SSCP patterns within the coding region and one in an intron of the VHL gene. SCLC line NCI-H1672 had a somatic mutation, G to A at nucleotide (nt) 530, leading to amino acid substitution (glycine to aspartic acid) compared to normal DNA from the same patient. Mesothelioma line NCI-H28 had T to A mutation at nt 479 leading to leucine to histidine amino acid change. We found one frequent polymorphism A (0.72) or G (0.28) at nt 19 resulting in either serine or glycine at this position, changes also found in normal peripheral blood cell DNA, often in a heterozygous state. In addition, we found single rare polymorphisms which did not alter the coding region including: C to G at nt 396, G to T at nt 843, and C to T change in an intron. These results suggest that the VHL gene is only rarely mutated in thoracic malignancies.

Base Sequence↗
PubMed1994

Examination of human tumors for rhoA mutations.

rhoA encodes a ras-related GTP-binding protein that is thought to play a role in cytoskeletal organization. Recent evidence has suggested both that rhoA could act either as a dominant oncogene, since transfection of both normal and activated rho genes confer a transformed phenotype on fibroblast cells in culture, or as a recessive tumor suppressor gene, by virtue, in part, of its chromosomal location at 3p21, a site deleted in many human malignancies. In either case, a role for rhoA in the oncogenesis of human tumors would be supported by the finding of rhoA mutations in tumors. We therefore examined human tumors and cell lines for mutations in the protein coding regions of rhoA by RNAase protection analysis. We first examined the expression of rhoA in renal cell carcinoma cell lines in which 3p21 was heterozygously deleted or retained. We found no evidence for rhoA mutations in these specimens. We also examined RNA from lung, breast, colon or ovarian tumors and also found no evidence of activating rhoA mutations. Furthermore, there was no relation between the level of rhoA mRNA expression and the presence or absence of 3p21 deletions in the renal cell carcinoma specimens. Thus, although rhoA has transforming potential in vitro, there is no evidence that it is activated by mutation in human malignancies, or that it could act as a tumor suppressor gene in tumors in which 3p21 is deleted.

Carcinoma, Renal Cell↗
PubMed1993

Chromosome 17p deletions and p53 mutations in renal cell carcinoma.

Studies of the role of tumor suppressor genes in human renal cell carcinoma from our laboratory have suggested the presence of a disease gene(s) on the short arm of chromosome 3. Little is known about the role other tumor suppressor genes may play in this malignancy. Abnormalities of chromosome 17p and, in particular of p53, are common in many human malignancies. In order to evaluate the role of this region in renal cell carcinoma, we performed restriction fragment length polymorphism analyses of chromosome 17 with probes localized to the p53 region. Fourteen of 29 (48%) evaluable cell lines showed loss of heterozygosity at this locus. Northern blot analysis did not detect a p53 transcript in 4 of 27 cell lines tested. In addition, we screened cell lines for p53 mutations using a polymerase chain reaction-single strand conformation polymorphism technique. Cell lines positive for mutations by this technique were then sequenced. Mutations were detected in 11 of 33 (33%) cell lines, including 8 derived from primary tumors and 3 derived from metastatic foci. Six of 9 (67%) patients with loss of heterozygosity demonstrated a mutation in the remaining allele, while only 1 of 8 (13%) without loss of heterozygosity had a mutation. Three of 3 (100%) cell lines derived from metastases had the same mutation as their matched primary cell line. Loss or mutation of p53 did not correlate either with loss of chromosome 3p or with histological subtype. These results suggest that, while the primary disease gene for kidney cancer appears to be on chromosome 3, abnormalities of p53 are common and may be involved in the progression of this malignancy.

Base Sequence↗
PubMed1993

Regional DNA hypermethylation at D17S5 precedes 17p structural changes in the progression of renal tumors.

In a preceding paper for brain tumors, we demonstrate a tight association between regional hypermethylation at locus D17S5 of chromosome 17p and allelic loss of this chromosome. Because 17p allelic losses occur at the earliest stages of brain tumors, the exact temporal relationship between this event and the hypermethylation could not be elucidated. In renal cancers, two linked structural changes on chromosome 17p, allelic loss and p53 gene mutations, generally occur late in progression. We now show that D17S5 hypermethylation is tightly coupled to both of these genetic changes in late stage renal tumors. However, the methylation change is the only one of the 17p abnormalities which occurs at a high incidence in early-stage renal cancers (hypermethylation, 50%; 17p allelic loss, 13%; p53 mutations, 0%). Our results firmly suggest that D17S5 regional hypermethylation precedes the appearance of the consistent 17p genetic changes in renal cancers, suggesting that this event either marks, or may even cause, chromatin changes which predispose to genetic instability.

Alleles↗
PubMed1993

Effect of suramin on the mitogenic response of the human prostate carcinoma cell line PC-3.

BACKGROUND: Suramin is an anthelmintic drug that recently has been shown to have clinical efficacy in the treatment of patients with some advanced malignancies, including prostate carcinoma. The current study was done to assess the effect of suramin at clinically relevant doses on the growth in culture of a human prostatic carcinoma cell line, PC-3. METHODS: The antiproliferative effect of varying doses of suramin on PC-3 was assessed. Northern blot analysis was done to assess the potential changes in genetic expression at different times after the initiation of treatment. RESULTS: Suramin inhibited the proliferation of PC-3 in a dose-related manner (concentration range, 30-300 microM). Compared with fetal calf serum 2%, when the cells were grown in fetal calf serum 10%, higher concentrations of suramin were required to inhibit tritiated thymidine incorporation. When grown in RPMI without supplement, the PC-3 cell number remained the same. When 100 microM suramin was included, the cell number decreased. By contrast, when RPMI was supplemented with insulin, transferrin, and selenium (ITS), PC-3 grew well. The inhibition of the proliferation of PC-3 cells by suramin was decreased when ITS were added to the cells grown under serum-free conditions. CONCLUSIONS: These results were consistent with the hypothesis that in vitro inhibition of the growth of PC-3 cells by suramin may be caused, at least in part, by the growth factor antagonism of the drug. In fetal calf serum 2%, the suramin inhibition was reversible after 3 days. If the treatment was extended to 6 days, however, the PC-3 cells were unable to recover. Cell-cycle analysis revealed that, after 6 days of treatment, there was a decrease in the number of cells in G1 that corresponded with an increased number of cells in G2/M. This suggested that critical antineoplastic events were occurring during this time. Molecular analysis did not detect any altered expression of actin, transforming growth factors alpha or beta, or histone compared with untreated control samples.

Blotting, Northern↗
PubMed1993

Molecular genetic studies of sporadic and familial renal cell carcinoma.

Renal cell carcinoma (RCC) occurs predominantly as a sporadic disease but has familial forms. Cytogenetic and DNA deletion analyses show that both sporadic and familial RCC tumors arise as the result of chromosomal deletions in the 3p13-p26 region. von Hippel-Lindau (VHL) disease is a hereditary, multifocal syndrome that includes, among several affected organs, multiple, bilateral renal cysts and RCC. Although the VHL disease gene maps to chromosome 3p25-p26, data indicate that the VHL and sporadic RCC disease genes are likely to be independent. The authors hypothesize that VHL disease manifestations are controlled by mutations in the VHL disease gene and that the onset of VHL-associated kidney cancer is due to a second mutation of the linked sporadic RCC gene.

Carcinoma, Renal Cell↗
PubMed1992

Interleukin (IL)-2 and IL-3 induce distinct but overlapping responses in murine IL-3-dependent 32D cells transduced with human IL-2 receptor beta chain: involvement of tyrosine kinase(s) other than p56lck.

We have established IL-3-dependent 32D myeloid progenitor cells stably expressing the human IL-2 receptor beta chain (IL-2R beta). Whereas parental 32D cells proliferated only in response to IL-3, the transduced cells also proliferated in response to IL-2. Transduced cells expressed high- and intermediate-affinity IL-2Rs, resulting from expression of human IL-2R beta and murine IL-2R alpha chain (IL-2R alpha). IL-2 induced phenotypic changes not induced by IL-3, including the upregulated expression of endogenous murine IL-2R alpha and IL-2R beta and an increase in cell size. Therefore, the transduced IL-2R beta was not merely coupling with the IL-3 signaling pathway. IL-3 augmented several IL-2-induced responses including the up-regulation of IL-2R alpha. Both IL-2- and IL-3-induced proliferation and IL-2 induced IL-2R alpha expression were inhibited by the tyrosine kinase inhibitor herbimycin A. Thus, both IL-2- and IL-3-mediated effects required tyrosine kinase activity. The identity of the tyrosine kinase(s) mediating the IL-2 signals in these cells is not known but cannot be p56lck, a tyrosine kinase found in T cells, since 32D-IL-2R beta cells do not express p56lck.

Animals↗
PubMed1991

The gene encoding the Act-2 cytokine. Genomic structure, HTLV-I/Tax responsiveness of 5' upstream sequences, and chromosomal localization.

Act-2 is a cytokine that belongs to a superfamily of structurally related proteins. Act-2 expression is rapidly induced in T cells, B cells, and monocytes upon mitogenic stimulation. The Act-2 genomic locus is on chromosome 17q. The exons and exon/intron splice junctions have been sequenced, as have the sequences upstream of exon 1. A classical TATA box is located immediately upstream of the transcription initiation site. The upstream sequences possess promoter activity and can be functionally activated after treatment of Jurkat T cells with phythohemagglutinin plus phorbol myristrate acetate. In addition, Act-2 promoter chloramphenicol acetyltransferase constructs are expressed in human T cell lymphotropic virus type I (HTLV-I)-infected MT-2 cells and in Jurkat cells which can be induced to express the transactivator gene (tax) product of HTLV-I.

Amino Acid Sequence↗