Identification of a candidate tumour suppressor gene, MMAC1, at chromosome 10q23.3 that is mutated in multiple advanced cancers.

Deletions involving regions of chromosome 10 occur in the vast majority (> 90%) of human glioblastoma multiformes. A region at chromosome 10q23-24 was implicated to contain a tumour suppressor gene and the identification of homozygous deletions in four glioma cell lines further refined the location. We have identified a gene, designated MMAC1, that spans these deletions and encodes a widely expressed 5.5-kb mRNA. The predicted MMAC1 protein contains sequence motifs with significant homology to the catalytic domain of protein phosphatases and to the cytoskeletal proteins, tensin and auxilin. MMAC1 coding-region mutations were observed in a number of glioma, prostate, kidney and breast carcinoma cell lines or tumour specimens. Our results identify a strong candidate tumour suppressor gene at chromosome 10q23.3, whose loss of function appears to be associated with the oncogenesis of multiple human cancers.

Growth control in cultured 3T3 fibroblasts. Assays of cell proliferation and demonstration of a growth inhibitory activity.

Treatment of sparse, proliferating cultures of 3T3 cells (target cells) with medium conditioned by exposure to density-inhibited 3T3 cultures resulted in an inhibition of growth and division in the target cells when compared to similar treatment with unconditioned medium (UCM). This differential effect of conditioned medium (CM) and UCM on target cells was demonstrated using three assay systems: (a) assessment of total cell number; (b) measurement of [3H]thymidine incorporated into acid-precipitable DNA; and (c) determination of the percentage of radioactively labeled nuclei in individual cells after incorporation of [3H]thymidine. The difference in the total incorporation of [3H]thymidine in CM-treated and UCM-treated cells was reflected by a difference in the percent of labeled cells. There was no differences in the average number of grains per labeled cell in the two cultures. Moreover, the inhibitory effect of the CM on target cell proliferation was reversible. Finally, this growth inhibitory activity can be collected in serum-free medium, precipitated by ammonium sulfate, and fractionated by gel filtration. In these purification procedures, the inhibitory activity was consistently found to be associated with the protein-containing fractions of the CM. No activity was found upon similar treatment with UCM. These results suggest that a system has been developed for the purification and molecular analysis of growth inhibitory factors that may mediate growth control in culture fibroblasts.

Growth control in cultured 3T3 fibroblasts II. Molecular properties of a fraction enriched in growth inhibitory activity.

Treatment of sparse, proliferating cultures of 3T3 cells with medium conditioned by exposure to density-inhibited 3T3 cultures resulted in an inhibition of growth and division in the target cells when compared to similar treatment with unconditioned medium. This growth inhibitory activity was fractionated by ammonium sulfate precipitation and gel filtration, yielding one fraction that was 35-fold enriched in specific activity. Analysis of the chemical and biological properties of this highly active fraction indicated that: (a) it is an endogenous cell product, synthesized by the 3T3 cells and shed into the medium; (b) it is a protein and its activity is sensitive to treatment with pronase; (c) the constituent polypeptide chains have molecular weights of 10,000 and 13,000; and (d) it is not cytotoxic and its effect on target cells are reversible. These results suggest that we have partially purified from conditioned medium an endogenous growth regulatory factor that may play a role in density-dependent inhibition of growth in cultured fibroblasts. We propose the term Fibroblast Growth Regulator to describe this class of molecules.

Characterization of p53 and p21 functional interactions in glioma cells en route to apoptosis.

BACKGROUND: Alterations of the p53 (also called TP53) gene are one of the most common abnormalities in gliomas. We have previously reported that restoration of wild-type p53 protein function in glioma cells results in programmed cell death (apoptosis). Since p53 functions are mediated by genes that directly control the tumor suppressor effect of the p53 protein, understanding the relationship between p53 and p53-related genes in glioma cells will aid in the design of more rational treatment strategies for brain tumors. PURPOSE: We conducted this study to examine the timing of the p53-mediated events preceding apoptosis. More specifically, we undertook this work to characterize the genetic and cell cycle-related factors that may increase the resistance of glioma cells to p53-induced apoptosis. METHODS: Two human glioma cell lines (U-251 MG and U-373 MG) that express mutant p53 protein and two (U-87 MG and EFC-2) that express wild-type p53 protein were used. Replication-deficient adenovirus was utilized as an expression vector to transfer exogenous p53 and p21 complementary DNAs into the glioma cells; control cells were infected with the viral expression vector alone. To monitor gene transfer and the expression of exogenous genes (as well as the expression of endogenous genes), we used western blot analyses and immunohistochemistry analyses. Flow cytometry studies of cellular DNA content were performed to determine the cell cycle phenotype of the glioma cells before and after treatment. RESULTS: p53-mediated apoptosis was preceded by elevation in the levels of the p21 (cell cycle-related) and Bax (apoptosis-related) proteins. In addition, cell cycle analyses showed that glioma cells were arrested in the G2 phase before undergoing cell death. Transfer of p21 induced a G2 block but did not induce apoptosis. Moreover, coexpression of p21 and p53 prevented glioma cells from undergoing apoptosis. Expression of exogenous p53 in wild-type p53 cells did not induce elevation of Bax levels, arrest in G2 phase, or apoptosis. CONCLUSIONS AND IMPLICATIONS: Our data confirmed the ability of wild-type p53 to induce apoptosis in p53 mutant glioma cells. In addition, our results document that p21 plays a role in protecting cells from p53-mediated programmed cell death and suggest that p53-mediated apoptosis and p21 induction may represent, at least in certain cases, opposite signals. Finally, our data suggest that over expression of p21 in gliomas may be related to resistance to treatments that induce apoptosis.

Monoclonal antibodies against cell-surface antigens of the metastatic rat 13762NF mammary adenocarcinoma and their cross-reactivity with human breast carcinomas.

Spleen cells from rats bearing syngeneic metastatic 13762NF mammary adenocarcinoma clone MTLn3 tumors were fused with the rat myeloma Y3 Ag1.2.3 to generate a panel of monoclonal antibodies (MAbs). The MAbs could be divided into three groups: those cross-reactive with all 13762NF cells; those reactive with cloned MTLn3 and MTC cells; and those predominantly reactive with the highly metastatic MTLn3 cells. One of these MAbs, MT10:21 (an immunoglobulin G2a), binds predominantly to highly metastatic MTLn3 cells and has a high tumor-cell affinity as determined by its saturation kinetics. MAb MT10:21 has a 6-h half-life on the MTLn3 cell surface and a 24-h half-life in the blood of syngeneic rats. Immunoblotting experiments using lysates from the cloned 13762NF sublines revealed that MAb MT10:21 binds to several proteins having relative molecular weights of 72,000, 73,000, and 120,000. Using an immunohistochemical procedure with frozen tissue sections, MAb MT10:21 shows little reactivity with normal rat mammary tissue, irrespective of the stage of the estrous cycle, and it failed to react with a number of other normal fetal and adult tissues. Furthermore, MAb MT10:21 is heterogeneous in its reactivity to cloned sublines of the 13762NF mammary adenocarcinoma, on both tissue cultured cells and tissue sections prepared from tumors growing in situ in the mammary fat pads of syngeneic rats. MAb MT10:21 reacted with certain human breast cancer cell lines and with a subpopulation of metastatic human breast cancer cells in frozen tissue sections from biopsies and autopsies. Metastases from breast cancers reacted more intensely than the primary tumors from which they were derived.

Expression of an altered epidermal growth factor receptor by human glioblastoma cells.

The expression and activities of epidermal growth factor (EGF) receptor and a highly related protein (Mr approximately 190,000 protein; p190) were characterized from a human glioma cell line, KE. p190 was specifically immunoprecipitated with a monoclonal antibody with specificity against the EGF receptor (Mr approximately 170,000; p170). Furthermore, both proteins were shown to possess tyrosine-protein kinase activities, although p170 required the presence of EGF to undergo autophosphorylation, whereas p190 appeared to be constitutively activated. Partial and total proteolytic polypeptide analyses of the two proteins suggested that their phosphopeptides were nearly identical and were phosphorylated on similar amino acid residues. However, a number of alterations were observed between [35S]methionine-labeled polypeptides of p170 and p190. This was also supported by the finding that the size of the protein cores of p170 and p190 was different. This observation is in agreement with Northern blot analysis in which KE cells expressed a novel EGF receptor RNA of 10.5 kilobases in addition to the previously reported 10-kilobase RNA. Southern blot analysis of the EGF receptor gene also revealed some amplification, approximately 4- to 5-fold; however, no significant rearrangements were noted in the KE cell DNA. These results suggest that p190 represents an endogeneous structurally and functionally altered EGF receptor.

Modulation of growth and epidermal growth factor receptor activity by retinoic acid in human glioma cells.

The growth-inhibitory activity of beta-all-trans-retinoic acid (RA) was examined on seven cultured human gliomas and cells derived from one normal brain. Response in monolayer cultures was heterogenous: three cell lines were completely resistant whereas five cell lines were growth inhibited with 50% inhibitory dose ranging from greater than 10(-5) to 1 x 10(-8) M. Two glioma cell lines capable of forming colonies in soft agar exhibited dose-dependent sensitivity to RA-induced growth inhibition, whereas another cell line was not affected by RA under either growth condition. Cell cycle analysis of the glial-derived cells has shown that the RA-sensitive cells accumulated in the G0-G1 phase. The cell surface expression of epidermal growth factor (EGF) receptors displayed by the various cells was either slightly increased or not affected by RA. In addition, the affinity of binding was slightly decreased in some sensitive cells. The activity of EGF receptor as assessed by immunocomplex-kinase assays revealed a dose-dependent decrease in autophosphorylation activity that appeared to correlate with the growth inhibition. The decrease in phosphokinase activity represented a dose-dependent inhibition of phosphorylation on tyrosine residues on EGF receptor as well as several other substrates. Furthermore, the autophosphorylation of either RA-treated or untreated EGF receptors occurred on similar amino acid residues. These results demonstrate that RA exhibits a heterogeneous growth-inhibitory activity against human glioma cells and suggest that the effects of RA may be mediated, at least in part, by modulation of EGF receptor phosphotyrosine kinase activity.

Elevated levels of M(r) 92,000 type IV collagenase in human brain tumors.

Local invasive growth is one of the key features of primary malignant brain tumors accompanied by remodeling of the vasculature and destruction of normal brain tissue. Tissue invasiveness is an essential biological function used by a tumor to overcome the various barriers to its progression. The expression of metalloproteases has been shown to play a critical role in the invasive process in a number of tumors; however, their expression in human brain tumors has not been previously reported. In this study we showed metalloprotease activities at M(r) 240,000, 123,000, 92,000, 72,000, and 67,000 in brain tumor extracts. These enzyme activities were inhibited by EDTA, an inhibitor of metalloproteases. Significant increases in levels of protease bands at M(r) 92,000, 123,000, and 240,000 were observed in glioblastoma and metastatic lung tumors. Enzymatic inhibition and Western blotting with M(r) 92,000 type IV collagenase antibody confirmed the presence of M(r) 92,000 type IV collagenase in all samples. Quantitative analysis by densitometry showed 8-10-fold and 6-8-fold increases in M(r) 92,000 type IV collagenase activity in glioblastoma and metastatic lung carcinoma samples, respectively, when compared with normal brain, meningioma, astrocytoma, metastatic colon, and breast carcinoma samples. These findings provide evidence for elevated levels of metalloproteases in glioblastomas and suggest a therapeutic target for minimizing the invasive propensity of gliomas using protease inhibitors.

Analysis of the functional role of chromosome 10 loss in human glioblastomas.

Molecular and cytogenetic analyses of primary brain tumors have shown that losses on chromosome 10 occur very frequently in human glioblastoma multiforme suggesting the presence of a glioma-associated tumor suppressor gene on this chromosome. To examine this hypothesis, a copy of chromosome 10 derived from a human fibroblast cell line was introduced into the human glioma cell line U251 by microcell-mediated chromosomal transfer. A human chromosome 2 was also independently introduced into U251 cells. The presence of novel chromosomes or chromosomal fragments was confirmed by molecular and karyotypic analyses. The hybrid clones containing a transferred chromosome 10 exhibited a suppression of their transformed and tumorigenic phenotype in vivo and in vitro, whereas cells containing a transferred chromosome 2 failed to alter their phenotype. The hybrid cells containing a transferred chromosome 10 displayed a significant decrease in their saturation density and an altered cellular morphology at high cell density but only a slight decrease in their exponential growth rate. A dramatic decrease was observed in the ability of cells with an introduced chromosome 10 to grow in soft agarose. The introduction of chromosome 10 completely suppressed tumor formation when the hybrid cells were injected into nude mice. These findings indicate that chromosome 10 harbors a tumor suppressor gene that is directly involved in glioma oncogenesis.

Altered expression and distribution of heparan sulfate proteoglycans in human gliomas.

The expression of heparan sulfate proteoglycans (HSPGs) by human glioma cells was examined by biochemical and immunological methods in vitro and in vivo. Chondroitin sulfate was shown to represent the major [3H]glucosamine-labeled glycosaminoglycan synthesized by cultured normal brain cells. However, high-grade glioma-derived cells were shown to express significantly increased quantities of hyaluronic acid and heparan sulfate and approximately equal amounts of chondroitin sulfate compared with normal glial cells. To investigate further the differential expression of HSPGs, proteoglycans were isolated from glioma cells and were used as an immunogen to generate monoclonal antibodies (MAbs). One of these MAbs, 39H (an IgM), was shown to bind more to high-grade glioma-derived cells then to low-grade glioma or normal brain cells in vitro. MAb 39H was also observed to bind to isolated HSPGs but not to heparan sulfate glycosaminoglycan chains or trypsin-treated cells. Immunofluorescence staining of the cultured high-grade glioma cells revealed an intense diffuse cell surface staining pattern over the entire cell and also isolated footpads. In contrast, the low-grade tumor or normal glial cells showed a distinctive punctated staining. A similar differential staining of MAb 39H was most prominent between tissue sections of glioblastoma multiforme and anaplastic astrocytomas versus low-grade astrocytomas and normal brain. The low grade gliomas exhibited a weak punctated staining, whereas the high-grade gliomas showed significantly more intense staining, particularly along the apical regions of the cells. These results suggest that altered expression of HSPGs may be related to the malignant transformation or growth potential of glial-derived cells.

Inhibition of angiogenesis in human glioblastomas by chromosome 10 induction of thrombospondin-1.

Glioblastoma multiforme is distinguished from its less malignant astrocytoma precursors by intense angiogenesis and frequent loss of tumor suppressor genes on chromosome 10. Here we link these traits by showing that when a wild-type chromosome 10 was returned to any of three human glioblastoma cell lines U251, U87, or LG11, they lost their ability to form tumors in nude mice and switched to an antiangiogenic phenotype, as measured by the inhibition of capillary endothelial cell migration and of corneal neovascularization. This change in angiogenesis was directly due to the increased secretion of a potent inhibitor of angiogenesis, thrombospondin-1, because: (a) neutralizing thrombospondin completely relieved the inhibition; (b) the inhibitory activity of thrombospondin was not dependent on transforming growth factor beta; and (c) chromosome 10 introduction did not alter secreted inducing activity. The inducing activity was dependent on vascular endothelial cell growth factor and had an ED50 of 10 microg/ml in media conditioned by parental cells and 9-13 microg/ml in media conditioned by chromosome 10 revertants. Normal human astrocytes were also antiangiogenic due to secreted thrombospondin. The effect of chromosome 10 on thrombospondin production in vitro was reflected in patient material. Normal brain and lower grade astrocytomas known to retain chromosome 10 stained strongly for thrombospondin, but 12 of 13 glioblastomas, the majority of which lose chromosome 10, did not. These data indicate that the loss of tumor suppressors on chromosome 10 contributes to the aggressive malignancy of glioblastomas in part by releasing constraints on angiogenesis that are maintained by thrombospondin in lower grade tumors.

Regulation of Akt/PKB activity, cellular growth, and apoptosis in prostate carcinoma cells by MMAC/PTEN.

Understanding the functional roles of the molecular alterations that are involved in the oncogenesis of prostate cancer, the second most frequent cause of cancer-related deaths among men in the United States is the focus of numerous investigations. To examine the possible significance of alterations associated with the tumor suppressor gene, MMAC/PTEN, in prostate carcinoma, the biological and biochemical effects of MMAC/PTEN expression were examined in LNCaP cells, which are devoid of a functional gene product. Acute expression of MMAC/PTEN via an adenoviral construct resulted in a dose-dependent and specific inhibition of Akt/PKB activation, consistent with the phosphatidylinositol phosphatase activity of MMAC/PTEN. MMAC/PTEN expression induced apoptosis in LNCaP cells, although to a lesser extent than that observed with p53 via an adenoviral construct. However, MMAC/PTEN expression produced a growth inhibition that was significantly greater than that achieved with p53. Overexpression of Bcl-2 in LNCaP cells blocked MMAC/PTEN- and p53-induced apoptosis but not the growth-suppressive effects of MMAC/ PTEN, suggesting that the growth regulatory effects of MMAC/PTEN involve multiple pathways. These studies further implicate the loss of MMAC/PTEN as a significant event in prostate cancer and suggest that reintroduction of MMAC/PTEN into deficient prostate cancer cells may have therapeutic implications.

Adenovirus-mediated transfer of the p53 gene produces rapid and generalized death of human glioma cells via apoptosis.

Wild-type p53 is involved in several aspects of cell cycle control and suppression of transformation, inducing either apoptosis or G1 block in cell cycle progression. Using a recombinant adenovirus containing the wild-type p53 cDNA, the biological effects of the newly expressed wild-type p53 protein were examined in six human glioma cell lines. Three cell lines (U-251 MG, U-373 MG, and A-172) expressed endogenous mutant p53, and the other three (U-87 MG, EFC-2, and D54 MG) expressed wild-type p53. The restoration of normal p53-encoded protein in the mutant cell lines induced apoptosis as assessed by morphological studies using nuclear staining, electron microscopy, and flow cytometric assays. In wild-type p53 cell lines, however, the overexpression of wild-type p53 did not result in apoptosis but inhibited cellular proliferation rather drastically and modified the neoplastic phenotype. Differential effects suggest two pathways for glioma oncogenesis and a possible therapeutic strategy.

Adenoviral transgene expression of MMAC/PTEN in human glioma cells inhibits Akt activation and induces anoikis.

The MMAC/PTEN tumor suppressor gene encodes for a phosphatase that recently has been shown to have phosphotidylinositol phosphatase activity, implicating its possible involvement in phosphatidylinositol 3'-kinase-mediated signaling. To investigate possible alterations in growth factor-mediated signal transduction, an adenovirus containing MMAC/PTEN, Ad-MMAC, previously shown to inhibit growth and tumorigenicity in glioma cells, was used to acutely express the transgene. Human glioma cells infected with Ad-MMAC but not with control adenoviruses exhibited an inhibition of phosphorylation of both activating residues of Akt, Ser-473, and Thr-308, along with Akt's serine/threonine kinase activity, without significantly altering Akt expression. The effects of functional MMAC/PTEN expression were relatively specific, because members of several other growth factor-mediated signaling pathways showed no altered responses. The presence of MMAC/PTEN also inhibited phosphorylation of BAD, although no evidence of apoptosis in the in situ treated cells was observed. However, U251 glioma cells infected with Ad-MMAC were induced to undergo anoikis at a significantly higher rate than U251 cels treated with control viruses or mock infected with media. These results demonstrate that the acute administration of MMAC/PTEN results in the inhibition of Akt-mediated signaling, growth inhibition, and anoikis, implying that loss of MMAC/PTEN increases cellular proliferation and significantly augments a cell's survival potential during cellular processes that are associated with malignancy.

Differential expression of MMAC/PTEN in glioblastoma multiforme: relationship to localization and prognosis.

MMAC/PTEN, a tumor suppressor gene located on chromosome 10q, has recently been shown to act as a phosphatidylinositol 3,4,5-triphosphate phosphatase and to modulate cell growth and apoptosis. Somatic mutations of MMAC/PTEN have been reported in a number of human cancers, especially in glioblastoma multiforme (GBM), although the number of identified mutations (approximately 10-35%) is significantly lower than the frequency of LOH affecting the MMAC/PTEN locus in the specimens (approximately 75-95%). To further investigate the possible alterations that may affect MMAC/PTEN, we examined the expression of the gene by reverse transcription-PCR in a series of gliomas. A significant difference (P < 0.001) was observed between the expression of MMAC/PTEN in GBMs versus lower grades of gliomas, thus mimicking the difference in allelic deletion associated with the locus in these tumors. Furthermore, Kaplan-Meier survival plots, adjusted for age and tumor grade, showed a significantly better prognosis for patients whose tumors expressed high levels of MMAC/PTEN. Additionally, immunostaining of GBMs revealed little or no MMAC/PTEN expression in about two-thirds of the tumors, whereas the other approximately one-third of tumors had significantly higher levels of expression. However, in about two-thirds of the high-expressing specimens, a heterogeneous pattern of expression was observed, indicating that certain cells within the tumor failed to express MMAC/PTEN. The combination of these results suggest that, in addition to molecular alterations affecting the gene, altered expression of MMAC/PTEN may play a significant role in the progression of GBM and patient outcome.

Antiangiogenesis treatment for gliomas: transfer of antisense-vascular endothelial growth factor inhibits tumor growth in vivo.

Presently, there is no effective treatment for glioblastoma, the most malignant and common brain tumor. Angiogenic factors are potentially optimal targets for therapeutic strategies because they are essential for tumor growth and progression. In this study, we sought a strategy for efficiently delivering an antisense cDNA molecule of the vascular endothelial growth factor (VEGF) to glioma cells. The recombinant adenoviral vector Ad5CMV-alphaVEGF carried the coding sequence of wild-type VEGF165 cDNA in an antisense orientation. Infection of U-87 MG malignant glioma cells with the Ad5CMV-alphaVEGF resulted in reduction of the level of the endogenous VEGF mRNA and drastically decreased the production of the targeted secretory form of the VEGF protein. Treatment of s.c. human glioma tumors established in nude mice with intralesional injection of Ad5CMV-alphaVEGF inhibited tumor growth. Taken together, these findings indicate that the efficient down-regulation of the VEGF produced by tumoral cells using antisense strategies has an antitumor effect in vivo. This is the first time that an adenoviral vector is used to transfer antisense VEGF sequence into glioma cells in an animal model, and our results suggest that this system may have clinical and therapeutic utility.

MMAC1/PTEN mutations in primary tumor specimens and tumor cell lines.

A candidate tumor suppressor gene, MMAC1/PTEN, located in human chromosome band 10q23, was recently identified based on sequence alterations observed in several glioma, breast, prostate, and kidney tumor specimens or cell lines. To further investigate the mutational profile of this gene in human cancers, we examined a large set of human tumor specimens and cancer cell lines of many types for 10q23 allelic losses and MMAC1 sequence alterations. Loss of heterozygosity (LOH) at the MMAC1 locus was observed in approximately one-half of the samples examined, consistent with the high frequency of 10q allelic loss reported for many cancers. Of 124 tumor specimens exhibiting LOH that have been screened for MMAC1 alterations to date, we have detected variants in 13 (approximately 10%) of these primary tumors; the highest frequency of variants was found in glioblastoma specimens (approximately 23%). Novel alterations identified in this gene include a missense variant in a melanoma sample and a splicing variant and a nonsense mutation in pediatric glioblastomas. Of 76 tumor cell lines prescreened for probable LOH, microsequence alterations of MMAC1 were detected in 12 (approximately 16%) of the lines, including those derived from astrocytoma, leukemia, and melanoma tumors, as well as bladder, breast, lung, prostate, submaxillary gland, and testis carcinomas. In addition, in this set of tumor cell lines, we detected 11 (approximately 14%) homozygous deletions that eliminated coding portions of MMAC1, a class of abnormality not detected by our methods in primary tumors. These data support the occurrence of inactivating MMAC1 alterations in multiple human cancer types. In addition, we report the discovery of a putative pseudogene of MMAC1 localized on chromosome 9.

MMAC1/PTEN inhibits cell growth and induces chemosensitivity to doxorubicin in human bladder cancer cells.

The development and progression of bladder cancer is associated with multiple alterations in the genome, including loss of chromosome 10. Recently, MMAC1/PTEN, a phosphatidylinositol phosphatase, has been mapped to chromosome 10q23. We previously demonstrated that MMAC1/PTEN has tumor suppressive properties in glioblastoma and prostate cancer. To investigate the efficacy of gene therapy with MMAC1/PTEN, we examined whether the exogenous introduction of MMAC1/PTEN via an adenoviral vector (Ad-MMAC) can inhibit tumor growth and reverse drug resistance to doxorubicin in human bladder cancer cells. Human bladder cancer cell lines UM-UC-3 and T24 were infected with Ad-MMAC to induce exogenous expression of MMAC1/PTEN. The cells were then analysed for cell growth and expression of phosphorylated protein kinase B (Akt/PKB) and MMAC1/PTEN. UM-UC-6dox, a doxorubicin resistant subline, was infected with Ad-MMAC to evaluate its role in reversing drug resistance to doxorubicin. We found that MMAC1/PTEN suppressed tumor growth in UM-UC-3 and T24 cells with arrest in the G1 phase of the cell cycle. We also showed that gene therapy with MMAC1/PTEN abrogated phosphorylated Akt/PKB expression in UM-UC-3, T24 and UMUC-6dox cells, and restored doxorubicin sensitivity in UM-UC-6dox. These data demonstrate that MMAC1/PTEN can induce growth suppression and increase sensitivity to doxorubicin in bladder cancer cells and suggest that the MMAC1/PTEN gene and its pathways can be therapeutic targets for bladder cancer.

Deletion mapping of chromosome 4 in head and neck squamous cell carcinoma.

Genomic deletions involving chromosome 4 have recently been implicated in several human cancers. To identify and characterize genetic events associated with the development of head and neck squamous cell carcinoma (HNSCC), a fine mapping of allelic losses associated with chromosome 4 was performed on DNA isolated from 27 matched primary tumor specimens and normal tissues. Loss of heterozygosity (LOH) of at least one chromosome 4 polymorphic allele was seen in the majority of tumors (92%). Allelic deletions were confined to short arm loci in four tumors and to the long arm loci in 12 tumors, suggesting the presence of two regions of common deletion. One region of frequent deletion was centered at D4S405 on 4p and included the loci D4S1546 to D4S428 in approximately 41% of the tumors. The common region of deletion on 4q was more complex and extended from D4S1571 to D4S1573. Frequent genetic alterations were observed within this region (4q25) and one marker, D4S407, exhibited a high frequency of LOH (>75%). These results indicate that alterations of chromosome 4 regions are associated with HNSCC tumorigenesis and further localizes the regions that may harbor tumor suppressor genes.

Identification of a novel gene product, RIG, that is down-regulated in human glioblastoma.

Genetic deletions to chromosome 10 have been extensively documented for human glioblastomas (GBMs). To identify gene products that may be involved in malignant progression, a subtractive hybridization was performed between GBM cells and hybrid cells suppressed for tumorigenicity following microcell transfer of chromosome 10. One novel cDNA isolated from this subtraction showed consistent upregulation (approximately 4 to 10-fold) that correlated with the nontumorigenic phenotype of the hybrid cells. Subsequent analysis resulted in the identification of a full length cDNA (2,569 bp) termed RIG (regulated in glioma). RIG expression was either not detected or detected only at low levels in cultured glioma cells and primary glioblastoma specimens compared to normal brain cells. The 2.6 kb RIG mRNA was expressed predominantly in normal brain with lower levels in heart and lung. Sequence analysis showed no significant homology to known gene products. Genomic alterations of RIG were present in approximately 25% of glioma cell lines examined. Also, RIG mapped to chromosome 11p15.1, a region that is known to be altered in malignant astrocytomas. The differential expression pattern, tissue distribution and chromosomal location of RIG suggests it serves as a molecular marker for or may play a role in the malignant progression of GBMs.