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Biomedical subjects

W K Cavenee

Publications and source records attributed to W K Cavenee.

At least 19 recordsLinked to original sources
PubMed1998

Cloning and characterization of three human forkhead genes that comprise an FKHR-like gene subfamily.

Alveolar rhabdomyosarcomas are associated with unique chromosomal translocations t(2;13) and t(1;13), which arise from fusion of the genes for the paired box proteins PAX3 and PAX7, respectively, to the FKHR (forkhead in rhabdomyosarcoma) gene on chromosome 13q14. Here we report the identification and characterization of three novel human forkhead genes with similarity to FKHR. The three genes (HGMW-approved symbols FKHRP1, FKHRL1, and FKHRL1P1) map to chromosomal regions 5q35.2-q35.3, 6q21, and 17p11, respectively. Based on amino acid sequence comparisons of their forkhead domains, FKHRL1, FKHRL1P1, and FKHRP1 share 86, 84, and 68% identity, respectively, with FKHR. While FKHR and FKHRL1 are expressed in every human adult tissue examined, FKHRP1 mRNA expression could not be detected, and FKHRL1P1 expression was present only at low levels. FKHR and FKHRL1 share a similar genomic organization, each having a very large intron 1 (FKHR approximately 130 kb and FKHRL1 > 90 kb), which bisects their respective forkhead domains at identical positions, as well as a second intron just downstream of each stop codon. FKHRP1 and FKHRL1P1 lack introns and contain stop codons that prevent them from yielding full-length proteins. Thus, while FKHR and FKHRL1 represent functional genes, FKHRP1 and FKHRL1P1 probably are processed pseudogenes. These results suggest that these four genes represent an FKHR-like gene subfamily within the larger human forkhead gene family.

Amino Acid Sequence↗
PubMed1997

Growth suppression of glioma cells by PTEN requires a functional phosphatase catalytic domain.

Deletions of all or part of chromosome 10 are the most common genetic alterations in high-grade gliomas. The PTEN gene (also called MMAC1 and TEP1) maps to chromosome region 10q23 and has been implicated as a target of alteration in gliomas and also in other cancers such as those of the breast, prostate, and kidney. Here we sought to provide a functional test of its candidacy as a growth suppressor in glioma cells. We used a combination of Northern blot analysis, protein truncation assays, and sequence analysis to determine the types and frequency of PTEN mutations in glioma cell lines so that we could define appropriate recipients to assess the growth suppressive function of PTEN by gene transfer. Introduction of wild-type PTEN into glioma cells containing endogenous mutant alleles caused growth suppression, but was without effect in cells containing endogenous wild-type PTEN. The ectopic expression of PTEN alleles, which carried mutations found in primary tumors and have been shown or are expected to inactivate its phosphatase activity, caused little growth suppression. These data strongly suggest that PTEN is a protein phosphatase that exhibits functional and specific growth-suppressing activity.

Brain Neoplasms↗
PubMed1997

Intracerebral tumor-associated hemorrhage caused by overexpression of the vascular endothelial growth factor isoforms VEGF121 and VEGF165 but not VEGF189.

The vascular endothelial growth factor (VEGF) has been shown to be a significant mediator of angiogenesis during a variety of normal and pathological processes, including tumor development. Human U87MG glioblastoma cells express the three VEGF isoforms: VEGF121, VEGF165, and VEGF189. Here, we have investigated whether these three isoforms have distinct roles in glioblastoma angiogenesis. Clones that overexpressed each isoform were derived and inoculated into mouse brains. Mice that received VEGF121- and VEGF165-overexpressing cells developed intracerebral hemorrhages after 60-90 hr. In contrast, mice implanted with VEGF189-overexpressing cells had only slightly larger tumors than those caused by parental cells and little evidence of hemorrhage at these early times after implantation, whereas, after longer periods of growth, enhanced angiogenicity and tumorigenicity were apparent. There was rapid blood vessel growth and breakdown around the tumors caused by cells overexpressing VEGF121 and VEGF165, whereas there was similar vascularization but no eruption in the vicinity of those tumors caused by cells overexpressing VEGF189, and none on the border of the tumors caused by the parental cells. Thus, by introducing VEGF-overexpressing glioblastoma cells into the brain, we have established a reproducible and predictable in vivo model of tumor-associated intracerebral hemorrhage caused by the enhanced expression of single molecular species. Such a model should be useful for uncovering the role of VEGF isoforms in the mechanisms of angiogenesis and for investigating intracerebral hemorrhage due to ischemic stroke or congenital malformations.

Adult↗
PubMed1997

Functional analysis of wild-type and malignant glioma derived CDKN2Abeta alleles: evidence for an RB-independent growth suppressive pathway.

The tumor suppressor gene CDKN2A (p16/MTS1/INK4A), which encodes the cyclin-dependent kinase inhibitor p16(INK4a), is a target of 9p21 deletions during the malignant progression of human gliomas. This gene also encodes a second protein product (human p16beta, murine p19ARF), which originates from an unrelated exon of CDKN2A (exon 1beta) spliced onto exon 2 in an alternate reading frame. Cell cycle arrest by p16beta is caused by an as yet unidentified pathway. In order to test the candidacy of p16beta as a glioma suppressor, we replaced p16(INK4a), p15(INK4b) and p16beta wild-type as well as a series of seven glioma-derived p16beta alleles (R87H, A112V, R120H, A121V, G125R, A128A and A128V), into glioma cell lines that had either CDKN2A-/RB+ (U-87MG and U-251MG) or CDKN2A+/RB- (LN-319) endogenous backgrounds and demonstrated that p16beta can act as a functional glioma cell growth suppressor. Moreover, p16beta, but not p16(INK4a) or p15(INK4b) inhibited the growth of RB-negative LN-319 cells, indicating that p16beta likely exerts its effects through an RB-independent pathway. In vitro and in vivo assays of pRB phosphorylation were consistent with this interpretation. Since none of the glioma-derived p16beta mutations inactivated their growth suppressive activities, it appears that mutations in CDKN2A exon 2 (which is shared in the coding sequences of p16(INK4a) and p16beta) likely exclusively target p16(INK4a).

Alleles↗
PubMed1997

Genetic alterations accumulate during cervical tumorigenesis and indicate a common origin for multifocal lesions.

Carcinomas of the uterine cervix are thought to arise from preinvasive dysplastic lesions, termed cervical intraepithelial neoplasias (CIN), grades I-III. Patients may present clinically with two or more distinct lesions of differing histological severity; however, the genesis of these multifocal lesions is unknown. Despite infection with high-risk human papilloma virus subtypes, which is a major etiological factor in disease pathogenesis, only a small and unpredictable number of dysplastic lesions progress to invasive cancer. Several lines of evidence suggest that additional somatic events, such as tumor suppressor gene inactivation, are required for malignant transformation. In support of this, loss of heterozygosity (LOH) analyses of invasive cervical carcinomas have identified several chromosomal arms likely to harbor tumor suppressor genes, of which regions on 3p, 4p, 4q, and 11q have been validated extensively. To evaluate the potential role of tumor suppressor gene inactivation in dysplastic progression, loci distributed on these four chromosomal regions were assessed for LOH in 42 CIN lesions of varying histological grade obtained from 17 patients. Analysis of at least 16 microsatellite loci in each lesion revealed allelic losses involving one or more of these chromosomal regions in 0% of CIN I lesions; 25% of CIN II lesions; and 88% of CIN III lesions, with 41% of CIN III lesions exhibiting LOH for three or more chromosomal regions. In addition, where LOH was scored for the same locus at a particular chromosomal region in all of the multiple lesions from a single patient, the same allele was lost at each locus, without exception. Statistical analysis of these allele-specific losses strongly suggests that topologically distinct lesions are related and likely arise from a common precursor cell.

Cell Transformation, Neoplastic↗
PubMed1997

High resolution analysis of chromosome 3p alterations in cervical carcinoma.

Although loss of heterozygosity (LOH) for loci on chromosome 3p is a common event in cervical carcinoma (CC), the frequency and affected regions of 3p are inconsistent among studies. Here we report a comprehensive analysis of LOH on 3p in 66 primary tumors and 16 CC-derived cell lines using a high density of marker loci. Clonal LOH was found in over 70% of primary tumors, and the patterns of loss indicated four to five target regions, with 3p14 being the most frequent. The majority of tumors had complex patterns of allelic imbalance, with regions of subclonal and clonal losses often present in individual tumors. We exploited marker homozygosity in CC-derived cell lines as an indirect measure of LOH and identified four homozygous deletions (HDs) during this analysis at loci located within the 3p14.2 region to which the FHIT gene has been mapped recently. This led to a careful reevaluation of the LOH patterns in primary CCs, which showed apparent retention of heterozygosity for loci in this region indicative of the presence of several additional HDs. To our knowledge, this is the first report of HDs encompassing the FHIT gene region in primary tumor samples and underscores the usefulness of high resolution genetic analysis of tumor genomes in determining the chromosomal aberrations underlying the malignant progression of CC.

Alleles↗
PubMed1997

The expression of RET and its multiple splice forms in developing human kidney.

A series of inductive events between two different cell groups, the ureteric bud epithelium and metanephric mesenchyme, gives rise to the functional mammalian kidney. These reciprocal inductive interactions involve a number of molecules, one of which is the RET receptor tyrosine kinase. The phenotype of mice lacking functional RET includes kidney agenesis or severe dysgenesis, indicating a requirement for RET in kidney organogenesis. To investigate RET expression in human kidney development, we used a semi-quantitative RT-PCR-based strategy to examine a panel of kidney RNA samples ranging from 8-24 weeks gestational age. We found RET expression was highest earlier in development (14 weeks) with expression decreasing through to 24 weeks gestation. While three alternative RET transcripts generated by exon skipping at the 5' end of the gene were all detected throughout kidney development, expression of one transcript, RET2/6, where exon 2 was spliced to exon 6, varied relative to full length RET during this period. Levels of RET2/6 were highest at the earliest age of fetal kidney examined (8 weeks) and decreased relative to all other RET transcripts to low adult levels. The period of high expression coincides with a period of rapid bud bifurcation. Thus, it is possible that RET2/6 has a role in the early growth and differentiation of the human kidney.

Alternative Splicing↗
PubMed1997

Cyclin-dependent kinase 6 (CDK6) amplification in human gliomas identified using two-dimensional separation of genomic DNA.

DNA amplification is a common mechanism invoked by many human tumors to elicit overexpression of genes whose products are involved in drug resistance or cell proliferation. Although amplified regions in tumor DNA may exceed several megabases in size, segments of amplicons with a high probability of containing gene sequences may be amenable to detection by restriction landmark genomic scanning (RLGS), a high-resolution DNA analysis that separates labeled NotI fragments in two dimensions. Here, we tested this by applying RLGS to matched samples of glioma and normal brain DNA and found tumor-specific amplification of the gene encoding cyclin-dependent kinase 6 (CDK6), an observation not previously reported in human tumors. The CDK6 gene has been localized to chromosome 7q21-22, but in the gliomas studied here, it was not coamplified with either the syntenic MET (7q31) or epidermal growth factor receptor (7p11-p12) genes, suggesting that this may be part of a novel amplicon in gliomas. We then corroborated this finding by identifying both amplification-associated and amplification-independent increases in CDK6 protein levels in gliomas relative to matched normal brain samples. These data implicate the CDK6 gene in genomic amplification and illustrate the potential of RLGS for the more general identification and cloning of novel genes that are amplified in human cancer.

Base Sequence↗
PubMed1997

Point mutations can inactivate in vitro and in vivo activities of p16(INK4a)/CDKN2A in human glioma.

Deletions of chromosomal region 9p21 are among the most common genetic alterations observed during the clonal evolution of high grade malignant gliomas. Structural and functional evidence has suggested that homozygous deletion involving CDKN2A (the genetic locus encoding the cyclin-dependent kinase inhibitor p16(NK4a)) is a mechanism of inactivation of this gene and that it can be a growth suppressor in human gliomas. However, the presence of other potential suppressor genes in the 9p21 region and the relatively large sizes of the deletions has made it difficult to be certain that the CDKN2A gene is their actual target. Here, we tested this hypothesis by determining the growth suppressive effects, cell cycle inhibitions, and the activities of seven naturally occurring glioma-derived CDKN2A alleles carrying point mutations and found that two of them were functionally compromised. To resolve discrepancies among the different existing functional assays, we developed an assay for p16(INK4a) function that allowed us to demonstrate that the expression of wild-type CDKN2A, but not alleles with inactivating mutations, prevents pRB phosphorylation in vivo in human glioma cells. These data suggest that CDKN2A is a critical target for mutational inactivation in human malignant gliomas.

Alleles↗
PubMed1997

The enhanced tumorigenic activity of a mutant epidermal growth factor receptor common in human cancers is mediated by threshold levels of constitutive tyrosine phosphorylation and unattenuated signaling.

Deregulation of signaling by the epidermal growth factor receptor (EGFR) is common in human malignancy progression. One mutant EGFR (variously named DeltaEGFR, de2-7 EGFR, or EGFRvIII), which occurs frequently in human cancers, lacks a portion of the extracellular ligand-binding domain due to genomic deletions that eliminate exons 2 to 7 and confers a dramatic enhancement of brain tumor cell tumorigenicity in vivo. In order to dissect the molecular mechanisms of this activity, we analyzed location, autophosphorylation, and attenuation of the mutant receptors. The mutant receptors were expressed on the cell surface and constitutively autophosphorylated at a significantly decreased level compared with wild-type EGFR activated by ligand treatment. Unlike wild-type EGFR, the constitutively active DeltaEGFR were not down-regulated, suggesting that the altered conformation of the mutant did not result in exposure of receptor sequence motifs required for endocytosis and lysosomal sorting. Mutational analysis showed that the enhanced tumorigenicity was dependent on intrinsic tyrosine kinase activity and was mediated through the carboxyl terminus. In contrast with wild-type receptor, mutation of any major tyrosine autophosphorylation site abolished these activities suggesting that the biological functions of DeltaEGFR are due to low constitutive activation with mitogenic effects amplified by failure to attenuate signaling by receptor down-regulation.

Amino Acid Sequence↗
PubMed1997

Expression and distribution of vascular endothelial growth factor protein in human brain tumors.

Marked neovascularization is a hallmark of many neoplasms in the nervous system. Recent reports indicate that the endothelial mitogen vascular endothelial growth factor (VEGF) may play a critical role in the regulation of vascular endothelial proliferation in malignant gliomas. Using novel monoclonal antibodies to the VEGF polypeptide we have determined the expression and cellular distribution of VEGF protein in a representative series of 171 human central nervous system (CNS) tumors by immunohistochemistry and immunoblotting. In agreement with previous in situ hybridization data, 19 out of 20 glioblastomas (95%) showed immunoreactivity for VEGF, whereas both the percentage of immunoreactive tumors and the extent of immunoreactivity for VEGF were significantly lower in astrocytomas. Of the pilocytic astrocytomas (WHO grade I) 44% were immunoreactive for VEGF, but we observed several cases with pronounced vascular proliferates in the absence of VEGF. In ependymomas, meningiomas, hemangioblastomas, and primitive neuroectodermal tumors, there was no correlation between VEGF expression, vascular endothelial proliferation and the grade of malignancy. Oligodendrogliomas and the oligodendroglial component of mixed gliomas lacked immunoreactive VEGF, indicating that endothelial growth factors other than VEGF may regulate tumor angiogenesis in these neoplasms. Western blot analysis showed a predominant VEGF protein species of 23 kDa and confirmed the immunohistochemical data in all cases. Our findings demonstrate that VEGF is expressed in a wide spectrum of brain tumors in which it may induce neovascularization. However, other angiogenic factors also appear to contribute to the vascularization of CNS neoplasms.

Astrocytoma↗
PubMed1997

Advances in the molecular genetics of gliomas.

Malignant gliomas are the most common primary tumors of the central nervous system but remain clinically intractable. This has engendered substantial efforts to elucidate the molecular genetic and biologic basis of glioma formation. This review focuses on recent discoveries of the genetic aberrations that occur during the progression of gliomas. These include those that inactivate tumor suppressor genes and activate oncogenes. The biologic consequences of such genetic changes on various tumor cell-host environment interactions are also described.

Apoptosis↗
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↗