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W K Cavenee

Publications and source records attributed to W K Cavenee.

At least 73 records · Page 4Linked to original sources
PubMed1996

A common mutant epidermal growth factor receptor confers enhanced tumorigenicity on human glioblastoma cells by increasing proliferation and reducing apoptosis.

Alterations of the EGFR gene occur frequently in human gliomas where the most common is an in-frame deletion of exons 2-7 from the extracellular domain, resulting in a truncated mutant receptor (deltaEGFR or de 2-7 EGFR). We previously demonstrated that introduction of deltaEGFR into human U87MG glioblastoma cells (U87MG.deltaEGFR) conferred remarkably enhanced tumorigenicity in vivo. Here, we show by cell-mixing experiments that the enhanced tumorigenicity conferred by deltaEGFR is attributable to a growth advantage intrinsic to cells expressing the mutant receptor. We analyzed the labeling index of the proliferation markers Ki-67 and bromodeoxyuridine and found that tumors derived from U87MG.deltaEGFR cells had significantly higher labeling indexes than those of tumors derived from U87MG cells that were either naive, expressed kinase-deficient mutants of deltaEGFR, or overexpressed exogenous wild-type EGFR. We also utilized terminal deoxynucleotidyl transferase-mediated nick end-labeling assays and showed that the apoptotic index of U87MG.deltaEGFR tumors was more than 4-fold lower than that of parental U87MG tumors. This decrease in cell death was inversely correlated with the expression level of Bcl-X(L), a negative regulator of apoptosis, which was more than 3-fold higher in U87MG.deltaEGFR-derived tumors than in those derived from parental cells. Similar observations were obtained in vitro in serum-free conditions. These results suggest that deltaEGFR exerts its pronounced enhancement of glioblastoma tumorigenicity by stimulating proliferation and inhibiting apoptosis and that the effects are directly attributable to its constitutively active signal.

Animals↗
PubMed1996

Enhanced tumorigenic behavior of glioblastoma cells expressing a truncated epidermal growth factor receptor is mediated through the Ras-Shc-Grb2 pathway.

A mutant epidermal growth factor receptor (DeltaEGFR) containing a deletion of 267 amino acids from the extracellular domain is common in human glioblastomas. We have previously shown that the mutant receptor fails to bind EGF, is constitutively phosphorylated, and confers upon U87MG glioblastoma cells expressing it (U87MG. DeltaEGFR), an increased ability to form tumors in mice. Here we demonstrate that the constitutively phosphorylated DeltaEGFR enhances growth of glioblastoma cells through increased activity of Ras: 1) there was an increase in the proportion of Ras present in the GTP-bound form, and 2) introduction of neutralizing anti-Ras 259 antibodies into U87MG and U87MG.DeltaEGFR cells by microinjection inhibited DNA synthesis to the same low level in both cell populations. We also show that the truncated EGF receptor constitutively associates with the adapter proteins Shc and Grb2 which are involved in the recruitment of Ras to activated receptors. Several derivatives of DeltaEGFR containing single, or multiple mutations at critical autophosphorylation sites were constructed and used to demonstrate that the major Shc binding site is Tyr-1148, and that Grb2 association occurs primarily through Tyr-1068. We conclude that the increased tumorigenic potential of glioblastoma cells expressing the truncated EGF receptor is due at least in part to Ras activation presumably involving the Shc and Grb2 adapter proteins.

Adaptor Proteins, Signal Transducing↗
PubMed1996

Tyrphostin AG 1478 preferentially inhibits human glioma cells expressing truncated rather than wild-type epidermal growth factor receptors.

The effects of a new epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, tyrphostin AG 1478, were tested on three related human glioma cell lines: U87MG, which expressed endogenous wild-type (wt) EGFR, and two retrovirally infected U87MG cell populations which over-expressed either wt (U87MG.wtEGFR) or truncated EGFR (U87MG. delta EGFR). Although AG 1478 inhibited cell growth, DNA synthesis, EGFR tyrosine kinase activity, and receptor autophosphorylation of each cell line in a dose-dependent manner, it was significantly more potent in U87MG. delta EGFR cells than in the other two cell lines. The increased inhibitory response of U87MG. delta EGFR cells was due to a greater sensitivity of the constitutively autophosphorylated Mr 140,000 and 155,000 delta EGFR species to AG 1478. These results suggest that AG 1478 is a relatively specific inhibitor of the delta EGFR, and this finding may have important therapeutic implications since the delta EGFR occurs frequently in glioblastomas and in breast, lung, and ovarian cancers.

Benzylidene Compounds↗
PubMed1996

cAMP effects on myogenic gene expression in rhabdomyosarcoma cells.

Rhabdomyosarcoma is a tumor of skeletal muscle origin affecting children and young adults. Although relatively undifferentiated, cell lines derived from this tumor express myogenic regulatory factors and so may be useful models of abortive myogenic differentiation. In the present studies, we have determined the effect of increased intracellular cAMP on proliferation, morphologic differentiation, and expression of myogenic genes in the prototypic embryonal rhabdomyosarcoma cell line, RD. Whereas growth in dibutyryl cAMP (dbcAMP), forskolin, or butyrate led to morphologic differentiation, growth in dbcAMP inhibited proliferation, while growth in butyrate slowed but did not stop cell division. Expression of the genes for myogenin and myosin light chain was inhibited by dbcAMP, while butyrate decreased myogenin and increased myosin light chain transcription. MyoD and MRF4 expression was not altered under either condition and no myf5 expression was detected. We also determined the effects of dbcAMP and butyrate on total protein expression, as well as on a panel of muscle- and neural-specific proteins using functional assays, immunohistochemistry, and immunoprecipitation. The total protein levels of cells treated with either agent were double those of untreated cells. DbcAMP increased the activity of acetylcholinesterase (AChE) up to 10-fold compared to untreated cells, while butyrate had a substantially lesser effect. These increases were due to increased AChE protein synthesis and stability in dbcAMP treated cells, compared to butyrate or untreated cells. Finally, cells under all conditions expressed MAP2, a neural-specific microtubule associated protein. Together, these data suggest that intracellular cAMP levels modulate distinct subsets of the myogenic differentiation pathway in rhabdomyosarcoma cells. Moreover, they also indicate that RD cells are able to express markers of different cell lineages, which may help explain some of the paradoxical features of these tumors.

Acetylcholinesterase↗
PubMed1996

Suppression of glioblastoma angiogenicity and tumorigenicity by inhibition of endogenous expression of vascular endothelial growth factor.

The development of new capillary networks from the normal microvasculature of the host appears to be required for growth of solid tumors. Tumor cells influence this process by producing both inhibitors and positive effectors of angiogenesis. Among the latter, the vascular endothelial growth factor (VEGF) has assumed prime candidacy as a major positive physiological effector. Here, we have directly tested this hypothesis in the brain tumor, glioblastoma multiforme, one of the most highly vascularized human cancers. We introduced an antisense VEGF expression construct into glioblastoma cells and found that (i) VEGF mRNA and protein levels were markedly reduced, (ii) the modified cells did not secrete sufficient factors so as to be chemoattractive for primary human microvascular endothelial cells, (iii) the modified cells were not able to sustain tumor growth in immunodeficient animals, and (iv) the density of in vivo blood vessel formation was reduced in direct relation to the reduction of VEGF secretion and tumor formation. Moreover, revertant cells that recovered the ability to secrete VEGF regained each of these tumorigenic properties. These results suggest that VEGF plays a major angiogenic role in glioblastoma.

Animals↗
PubMed1996

Simultaneous assessment of loss of heterozygosity at multiple microsatellite loci using semi-automated fluorescence-based detection: subregional mapping of chromosome 4 in cervical carcinoma.

Detection of loss of heterozygosity (LOH) by comparison of normal and tumor genotypes using PCR-based microsatellite loci provides considerable advantages over traditional Southern blotting-based approaches. However, current methodologies are limited by several factors, including the numbers of loci that can be evaluated for LOH in a single experiment, the discrimination of true alleles versus "stutter bands," and the use of radionucleotides in detecting PCR products. Here we describe methods for high throughput simultaneous assessment of LOH at multiple loci in human tumors; these methods rely on the detection of amplified microsatellite loci by fluorescence-based DNA sequencing technology. Data generated by this approach are processed by several computer software programs that enable the automated linear quantitation and calculation of allelic ratios, allowing rapid ascertainment of LOH. As a test of this approach, genotypes at a series of loci on chromosome 4 were determined for 58 carcinomas of the uterine cervix. The results underscore the efficacy, sensitivity, and remarkable reproducibility of this approach to LOH detection and provide subchromosomal localization of two regions of chromosome 4 commonly altered in cervical tumors.

Chromosome Deletion↗
PubMed1996

Silencing of p16/CDKN2 expression in human gliomas by methylation and chromatin condensation.

The product of the p16/CDKN2 locus, p16ink4, negatively regulates the cell cycle through binding and inactivation of cyclin-dependent kinases (CDKs) 4 and 6. This locus is frequently targeted for deletion in cell lines and primary tumor tissues. In gliomas, although up to 50% do not have detectable expression of p16/CDKN2 protein or mRNA, often the gene is wild type in sequence. Here, we tested the hypothesis that transcriptional repression of p16/CDKN2 in gliomas may be mediated by aberrant methylation of the CpG island, which is in the 5' region of the locus. Partial rather than complete p16/CDKN2 methylation was detected in 24% (10 of 42) of the gliomas, regardless of tumor grade, but was not observed in normal brain (0 of 10). We tested whether this partial methylation could inhibit expression in a human tumor cell line in which suppressed p16/CDKN2 expression was associated with both methylation and tightly compacted chromatin around the p16/CDKN2 promoter. Exposure of these cells to 5-aza-2-deoxycytidine resulted in a dramatic increase in promoter accessibility and induction of p16/CDKN2 expression, indicating that chromatin structure, CpG island methylation, and p16/CDKN2 expression are intimately associated. Taken together, these data suggest that methylation occurs in only a subset of cells within gliomas and that the methylation-associated inactivation of p16/CDKN2 expression observed in many common human cancers may mechanistically result from structural changes in the chromatin containing the p16/CDKN2 locus.

Astrocytoma↗
PubMed1996

Analysis of replication error (RER+) phenotypes in cervical carcinoma.

Infection of epithelial cells with human papillomavirus is an important early event in the development of cervical dysplasia. However, progression to overt malignancy appears dependent upon further genetic and/or epigenetic events. We have recently developed methodologies for the simultaneous analysis of loss of heterozygosity (LOH) at multiple PCR-based microsatellite loci using semiautomated fluorescent DNA sequencing technology to determine the locations of tumor suppressor genes which are inactivated during tumor progression. While examining 30 microsatellite loci for LOH on chromosomes 3p, 4, and 11q, we detected novel tumor-specific alleles indicative of microsatellite instability (MI). The methodology allowed rapid and accurate comparison of over 3000 genotypes from 89 primary tumors and 10 cervical carcinoma-derived cell lines and showed that five tumors (5.6%) and one human papillomavirus-negative cell line, C33A, had genetic features consistent with a replication error (RER+) phenotype as defined by MI at two or more loci. In each of the RER+ tumors, LOH was also observed at one or more loci on each of the three chromosomes examined. These findings suggest that defects in DNA repair-associated genes are rarely acquired and do not supersede allelic loss during cervical carcinogenesis. In addition, the semiautomated multiplex approach has proven unequivocal in the detection and interpretation of MI and should greatly accelerate the rapidity and accuracy of analysis of such defects in tumors. Moreover, the number of loci that can be relatively easily examined in this way will also allow a detailed statistical consideration of the importance of such events.

Chromosomes, Human, Pair 11↗
PubMed1996

Detection of the t(2;13) chromosomal translocation in alveolar rhabdomyosarcoma using the reverse transcriptase-polymerase chain reaction.

Rhabdomyosarcoma, a small-, round-cell tumor of skeletal muscle, is the most common soft tissue sarcoma found in children. A specific and unique chromosomal translocation, t(2;13)(q35;q14), has been described cytogenetically in a subset of these tumors and is most often associated with the alveolar histologic subtype. The cloning and sequencing of complementary DNA from fusion transcripts expressed by both cell lines and tumors have shown that this chromosomal translocation results in the fusion of the PAX3 gene on chromosome 2 with a member of the forkhead gene family, FKHR, on chromosome 13. To detect this genetic abnormality we have developed a sensitive method which relies on a reverse transcriptase-polymerase chain reaction with primers designed to be specific for the chromosome 2 and chromosome 13 sides of the translocation. The utility of this approach was tested by analyzing a series of rhabdomyosarcoma cell lines and tumor samples. The data demonstrate that the transcripts derived from the t(2;13) were restricted to tumors having features of the alveolar subtype and that they could be detected with greater ease and sensitivity than with cytogenetic analysis. This approach will facilitate a large-scale group effort to determine the frequency as well as the prognostic and diagnostic significance of this chromosomal rearrangement.

Cell Line↗
PubMed1996

A common region of loss of heterozygosity in Wilms' tumor and embryonal rhabdomyosarcoma distal to the D11S988 locus on chromosome 11p15.5.

The development of Wilms' tumor has been associated with two genetic loci on chromosome 11: WT1 in 11p13 and WT2 in 11p15.5. Here, we have used loss of heterozygosity (LOH) in Wilms' tumors to narrow the WT2 locus distal to the D11S988 locus. A similar region was apparent for the clinically associated tumor, embryonal rhabdomyosarcoma. We have also demonstrated that a constitutional chromosome translocation breakpoint associated with Beckwith-Wiedemann syndrome and an acquired somatic chromosome translocation breakpoint in a rhabdoid tumor each occur in the same chromosomal interval as the smallest region of LOH in Wilms' tumors and embryonal rhabdomyosarcoma. Finally, we report the first Wilms' tumor without a cytogenetic deletion that shows targeted LOH for 11p15 and 11p13 while maintaining germline status for 11p14.

Base Sequence↗
PubMed1996

Population variation at the polymorphic ApaLI restriction enzyme site in intron 5 of the WT1 gene.

We examined 63 unrelated individuals from the United States for the Apa-LI polymorphism in intron 5 of the WT1 gene. Allele frequencies of 0.13 and 0.87 for the A and B alleles, respectively, and a heterozygosity index of 24% contrast sharply with previous data obtained in the Japanese population where allele frequencies of 0.55 and 0.45 for the A and B alleles and a heterozygosity index of 59% were reported. These data suggest genetic heterogeneity at the WT1 locus, which may contribute to the differences in the incidence of Wilms tumor between the two population groups.

Alleles↗
PubMed1996

Molecular biology of malignant degeneration of astrocytoma.

Cancer has been proposed to develop by a process of stepwise accumulation of growth-advantageous genetic alterations which result in the evolution of clones which are outgrowths of such rare cells [1]. This model has recently been extensively tested in human gliomas, the most common primary tumor of the adult central nervous system. Temporal disease progression involves an interplay between growth-suppressing and growth-promoting genes. Specifically for gliomas, genetic studies have indicated loss of germline heterozygosity for chromosome 17p; mutation of the p53 gene; overexpression of the platelet-derived growth factor-alpha receptor; allelic losses of chromosomes 22q, 13q, and 19q; deletion of the interferon-alpha and beta and CDKN2 loci on chromosome 9p; amplification and rearrangement of the epidermal growth factor receptor gene, and monosomy of chromosome 10. The following discussion details these genetic alterations and their consequences for the biology of glioma progression with the ultimate aim of providing new avenues for clinical intervention.

Adult↗
PubMed1995

EGFR gene amplification--rearrangement in human glioblastomas.

Immunostaining using an affinity-purified rabbit polyclonal antibody against the extracellular domain of the epidermal-growth-factor receptor (EGFR) showed over-expression occurring in a fraction of tumor cells in 17 out of 18 human glioblastomas and in a majority of cells in 7 of the 18. Southern-blotting technique using a full-length EGFR cDNA probe showed a variable degree of amplification in 10 of the 17 glioblastomas, which was associated with EGFR over-expression in each case. In 2 of the glioblastomas with EGFR gene amplification, a rearrangement of the gene affecting the extracellular domain of the receptor was identified and DNA sequence analyses revealed an identical deletion-rearrangement of 801 base pairs between exons 2 to 7, resulting in an in-frame fusion of exons 1 and 8.

Base Sequence↗
PubMed1995

Definition and refinement of chromosome 11 regions of loss of heterozygosity in breast cancer: identification of a new region at 11q23.3.

Chromosome 11 is frequently altered in several types of human neoplasms. In breast cancer, loss of heterozygosity has been described in two regions of this chromosome, 11p15 and 11q22-23. In this report we have dissected the two regions using high-density polymorphic markers, and have found that there are at least two independent areas of loss of heterozygosity in each region, suggesting that multiple genes on chromosome 11 may be targets of genetic alteration during tumor establishment or progression. The regions defined are: at 11p15, between loci D11S576 and D11S1318 and between D11S988 and D11S1318; at 11q23, between D11S2000 and D11S897 and between D11S528 and D11S990. The narrowing of these regions of loss should facilitate the cloning of the regions in yeast artificial chromosomes to identify the critical tumor suppressor genes.

Base Sequence↗
PubMed1995

Loss of wild-type p53 bestows a growth advantage on primary cortical astrocytes and facilitates their in vitro transformation.

Primary cortical astrocytes were isolated from normal (+/+), heterozygous (+/-), or homozygous (-/-) p53-knockout mice. The normal astrocytes grew slowly and underwent crisis after limited division, while the homozygously defective cells grew rapidly and without contact inhibition. These -/- cells could not initially form colonies in soft agarose but acquired this capability after 10 passages in FCS or basic fibroblast growth factor but not epidermal growth factor. Almost all -/- astrocytes weakly expressed glial fibrillary acidic protein at passage 10 and were also A2B5+ when cultured in basic fibroblast growth factor. Most heterozygous cells resembled normal ones; however, some survived crisis, grew rapidly, and formed colonies. Outgrowing cells had all lost the wild-type p53 allele. These molecular and cellular events mimic the early stages of human brain tumors, suggest a role for p53 in the earliest stages of disease progression, and provide an experimental system to analyze the effects of other tumor-specific mutations in the disease process.

Animals↗
PubMed1995

A defined region of loss of heterozygosity at 11q23 in cutaneous malignant melanoma.

Karyotypic and molecular data indicate that genetic alterations of the long arm of chromosome 11 (11q) may be involved in malignant melanoma. To test this we analyzed 5 polymorphic microsatellite repeats on 11q using a PCR-based assay for loss of heterozygosity in normal and tumor tissues from 24 individuals with cutaneous malignant melanoma of various stages. Our findings indicate that a tumor suppressor gene that plays a role in malignant melanoma is located on the long arm of chromosome 11, likely within a 51 cM region at 11q23. Its loss appears to be a late event in tumor progression and may serve as an indicator for a less favorable clinical outcome.

Adult↗
PubMed1995

Loss of P16INK4 expression is frequent in high grade gliomas.

P16INK4 is a cell cycle regulator that specifically binds to and inactivates cyclin-dependent kinase 4 (CDK4). Its encoding gene (p16/CDKN2) maps to chromosome 9p21, a region that undergoes frequent loss of heterozygosity in a variety of human tumors. We have analyzed the p16/CDKN2 gene and its expression in a series of primary glioma samples. Although homozygous deletion or mutation of the p16/CDKN2 gene was uncommon in this series and P16INK4 protein was detectable in all grade II tumors, it was present in only 50% of grade III and grade IV samples. Conversely, in some grade IV tumors that level of P16INK4 protein was elevated; in these cases, its target, CDK4, was amplified and overexpressed. These results suggest: (a) the involvement of P16INK4 in glioma progression; (b) that mechanisms other than mutation or deletion can down-regulate expression of the p16/CDKN2 gene; and (c) that the balance between CDK4 and its cognate inhibitor, P16INK4, may confer a cell growth advantage and facilitate tumor progression.

Astrocytoma↗