Robert Bookstein - Academia.edu (original) (raw)

Papers by Robert Bookstein

Cancer Research, May 15, 1999

Genetic alterations in the MMAC1 tumor suppressor gene (also referred to as PTEN or TEP1) occur i... more Genetic alterations in the MMAC1 tumor suppressor gene (also referred to as PTEN or TEP1) occur in several types of human cancers including glioblastoma. Growth suppression induced by overexpression of MMAC1 in cells with mutant MMAC1 alleles is thought to be mediated by the inhibition of signaling through the phosphatidylinositol 3-kinase pathway. However, the exact biochemical mechanisms by which MMAC1 exerts its growth-inhibitory effects are still unknown. Here we report that recombinant adenovirus-mediated overexpression of MMAC1 in three different MMAC1-mutant glioblastoma cell lines blocked progression from G0/G1 to S phase of the cell cycle. Cell cycle arrest correlated with the recruitment of the cyclin-dependent kinase (CDK) inhibitor, p27Kip1, to cyclin E immunocomplexes, which resulted in a reduction in CDK2 kinase activities and a decrease in levels of endogenous phosphorylated retinoblastoma protein. CDK4 kinase activities were unaffected, as were the levels of the CDK ...

The MMAC/PTEN tumor suppressor gene encodes for a phosphatase that recently has been shown to hav... more 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 ac tivity, without significantly altering Akt expression. The effects of func tional 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 phospho rylation of ...

Cancer research, 2002

Recent work identifies the AKT kinase as a potential mediator of tumor expansion in multiple myel... more Recent work identifies the AKT kinase as a potential mediator of tumor expansion in multiple myeloma. The finding of PTEN mutations in several myeloma cell lines suggests that loss of PTEN function may be one mechanism by which AKT activity is increased in this disease. Because PTEN-deficient myeloma cells may have up-regulated activity of the mammalian target of rapamycin (mTOR), downstream of AKT, they may be particularly sensitive to mTOR inhibition. To test this hypothesis, we challenged myeloma cell lines with CCI-779, a newly developed analogue of rapamycin and an efficient inhibitor of mTOR. Three of four PTEN-deficient cell lines with constitutively active AKT were remarkably sensitive to cytoreduction and G(1) arrest induced by CCI-779 with ID(50) concentrations of <1 nM. In contrast, myeloma cells expressing wild-type PTEN were >1000-fold more resistant. Acute expression of a constitutively active AKT gene in CCI-779-resistant myeloma cells containing wild-type PTEN ...

A candidate tumor suppressor gene, MMACJ/PTEN,located in human chromosome band 10q23, was recenti... more A candidate tumor suppressor gene, MMACJ/PTEN,located in human chromosome band 10q23, was recentiy identified based on sequence al terations 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 sped mens and cancer cell lines of many types for 10q23 allelic losses and MMACJ 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 lOq allelic loss reported for many cancers. Of 124 tumor spedmens exhibiting LOH that have been screened for MMAC1 alterations to date, we have detected variants in 13 (—10%)of these primary tumors; the highest frequency of variants was found in glioblastoma specimens (—23%). Novel alterations identified in this gene include a missense variant In a melanoma sample and a splicing variant and a...

Oncology and Therapy

Introduction: Next-generation sequencing has emerged as a clinical tool for the identification of... more Introduction: Next-generation sequencing has emerged as a clinical tool for the identification of actionable mutations to triage advanced colorectal cancer patients for targeted therapies. The literature is conflicted as to whether primaries or their metastases should be selected for sequencing. Some authors suggest that either site may be sequenced, whereas others recommend sequencing the primary, the metastasis, or even both tumors. Here, we address this issue head on with a meta-analysis and provide for the first time a set of sensible recommendations to make this determination. Methods: From our own series, we include 43 tumors from 13 patients including 14 primaries, 10 regional lymph node metastases, 17 distant metastases, and two anastomotic recurrences sequenced using the 50 gene Ion AmpliSeq cancer NGS panel v2. Results: Based on our new cohort and a metaanalysis, we found that * 77% of patient-matched primary-metastatic pairs have identical alterations in these 50 cancer-associated genes. Conclusions: Low tumor cellularity, tumor heterogeneity, clonal evolution, treatment status, sample quality, and/or size of the sequencing panel accounted for a proportion of the differential detection of mutations at primary and metastatic sites. The therapeutic implications of the most frequently discordant alterations (TP53, APC, PIK3CA, and SMAD4) are discussed. Our meta-analysis indicates that a subset of patients who fail initial therapy may benefit from sequencing of additional sites to identify new actionable genomic abnormalities not present in the initial analysis. Evidencebased recommendations are proposed.

Cancer Research, Jun 1, 1998

Mutated in multiple advanced cancers 1/phosphatase and tensin homo logue (MMACl/PTEN) is a novel ... more Mutated in multiple advanced cancers 1/phosphatase and tensin homo logue (MMACl/PTEN) is a novel tumor suppressor gene candidate located on chromosome 10 that is commonly mutated in human glioblastoma multiforme and several other cancer types. To evaluate the function of this gene as a tumor suppressor, we constructed a replication-defective adenovirus (MMCB) for efficient, transient transduction of MMAC1 into tumor cells. Infection of MMACl-mutated U87MG glioblastoma cells with MMCB resulted in dose-dependent exogenous MMAC1 protein expres sion as detected by Western blotting of cell lysates. In vitro proliferation of U87MG cells was inhibited by MMCB in comparison to several control adenoviruses at equal viral doses, implying a specific effect of MMAC1 expression. Anchorage-independent growth in soft agar was also inhibited by MMCB compared to control adenovirus. Tumorigenicity in nude mice of transiently transduced mass cell cultures was then assessed. MMCBinfected U87MG cells were almost completely nontumorigenic compared to untreated and several control adenovirus-treated cells at equal viral doses. These data support an in vivo tumor suppression activity of MMACl/PTEN and suggest that in vivo gene transfer with this recombi nant adenoviral vector has a potential use in cancer gene therapy.

Cancer Research, May 1, 2003

Approximately 30-40% of malignant glial tumors exhibit mutations in the tumor suppressor gene, PT... more Approximately 30-40% of malignant glial tumors exhibit mutations in the tumor suppressor gene, PTEN/MMAC. Additionally, these tumors are associated with (a) mutations in epidermal growth factor receptor (EGFR), leading to a pro-oncogenic constitutive activation, as well as amplification of its gene, and/or (b) mutations in p53, disrupting normal cellular homeostatic processes. Whereas PTEN/MMAC has been shown to possess antiangiogenic action, constitutively active EGFR or p53 gene defects have been associated with proangiogenic action. In this article, we asked if PTEN/MMAC gene transfer into human glioma cells that possess inactivating mutations of the PTEN/MMAC gene but also express either constitutively active EGFR (U87⌬EGFR cells) or possess an inactivating mutation of p53 (U251 cells) still display inhibited angiogenesis in orthotopic and ectopic models of gliomas. Human glioma xenografts treated with PTEN/MMAC gene transfer exhibited significantly decreased vascularity both in an orthotopic and in an ectopic model. Taken in combination, these results provide strong evidence of PTEN/ MMAC's role in regulating glioma angiogenesis even in the presence of strong proangiogenic signals provided by constitutive EGFR activation or p53 inactivation.

Cancer Research, May 15, 1999

Genetic alterations in the MMAC1 tumor suppressor gene (also referred to as PTEN or TEP1) occur i... more Genetic alterations in the MMAC1 tumor suppressor gene (also referred to as PTEN or TEP1) occur in several types of human cancers including glioblastoma. Growth suppression induced by overexpression of MMAC1 in cells with mutant MMAC1 alleles is thought to be mediated by the inhibition of signaling through the phosphatidylinositol 3-kinase pathway. However, the exact biochemical mechanisms by which MMAC1 exerts its growth-inhibitory effects are still unknown. Here we report that recombinant adenovirus-mediated overexpression of MMAC1 in three different MMAC1-mutant glioblastoma cell lines blocked progression from G 0 /G 1 to S phase of the cell cycle. Cell cycle arrest correlated with the recruitment of the cyclin-dependent kinase (CDK) inhibitor, p27 Kip1 , to cyclin E immunocomplexes, which resulted in a reduction in CDK2 kinase activities and a decrease in levels of endogenous phosphorylated retinoblastoma protein. CDK4 kinase activities were unaffected, as were the levels of the CDK inhibitor p21 Cip1 present in cyclin E immunocomplexes. Therefore, overexpression of MMAC1 via adenovirus-mediated gene transfer suppresses tumor cell growth through cell cycle inhibitory mechanisms, and as such, represents a potential therapeutic approach to treating glioblastomas.

Cancer Research, Nov 1, 2002

Smad proteins transduce signals carried by the transforming growth factor ␤ (TGF-␤) cytokine supe... more Smad proteins transduce signals carried by the transforming growth factor ␤ (TGF-␤) cytokine superfamily from receptor serine/threonine kinases at the cell surface to the nucleus, thereby affecting cell proliferation, differentiation, as well as pattern formation during early vertebrate development. Smad4/DPC4, located at chromosome 18q21, was identified as a candidate tumor suppressor gene that is inactivated in nearly half of all pancreatic carcinomas. For functional characterization of Smad4, a recombinant adenovirus encoding Smad4 (Ad-Smad4) was generated. When Smad4 was expressed in Smad4-null breast carcinoma cell line MDA-MB-468 using the recombinant adenovirus, TGF-␤ signaling was restored as determined by TGF-␤-dependent activity of plasminogen activator inhibitor 1 promoter and p21 expression. Infection with Ad-Smad4 in the presence of TGF-␤1 also resulted in an altered cell morphology that coincided with enhanced ␤1 integrin expression and reduced efficiency of colony formation in soft agar. In agreement with increased p21 expression, Smad4-expressing cells showed modest reduction in S phase. However, Smad4 expression did not lead to induction of apoptosis under normal culture conditions. Interestingly, when Smad4-expressing cells were detached and incubated in suspension, they underwent rapid apoptosis in a TGF-␤-dependent manner. Induction of apoptosis caused by loss of anchorage is known as anoikis. Anoikis is believed to prevent colonization elsewhere of detached cells. Additional characterization revealed an increase in the level of focal adhesion kinase 2 (or Pyk2) and activation of caspases 2, 3, 6, and 8 during anoikis because of Smad4 expression and restoration of TGF-␤ signaling. Because resistance to anoikis in tumor cells is thought to contribute to metastasis, our data suggest a functional basis for the strong correlation between defects in Smad4 and development of malignancy.

Journal of Cellular Biochemistry Supplement, Feb 1, 1994

Mutations of tumor suppressor genes are critical genetic alterations occurring during the genesis... more Mutations of tumor suppressor genes are critical genetic alterations occurring during the genesis and progression of human cancer, and consequently are candidates for use as surrogate endpoint biomarkers. The two most intensively studied suppressor genes, retinoblastoma (Rb) and p53, are mutated in approximately 20-50% of advanced-stage prostate cancers, but only rarely in early tumors. The precise DNA base changes, especially those affecting p53, may yield clues to relevant carcinogenic mechanisms. Increased expression of p53 in neoplastic cells, as detected by immunohistochemistry, may indicate mutation or a physiological response to DNA damage. Allelic losses of chromosome arms 8p and 16q are relatively common even in early prostate cancers. Quantitative measurement of allelic imbalance can be performed in preneoplastic or small neoplastic lesions, albeit with some technical challenge. The significance of whole-genome or regional allelic imbalance at various stages of prostatic oncogenesis has not been established.

Cancer Research

Recent work identifies the AKT kinase as a potential mediator of tumor expansion in multiple myel... more Recent work identifies the AKT kinase as a potential mediator of tumor expansion in multiple myeloma. The finding of PTEN mutations in several myeloma cell lines suggests that loss of PTEN function may be one mechanism by which AKT activity is increased in this disease. Because PTEN-deficient myeloma cells may have up-regulated activity of the mammalian target of rapamycin (mTOR), downstream of AKT, they may be particularly sensitive to mTOR inhibition. To test this hypothesis, we challenged myeloma cell lines with CCI-779, a newly developed analogue of rapamycin and an efficient inhibitor of mTOR. Three of four PTEN-deficient cell lines with constitutively active AKT were remarkably sensitive to cytoreduction and G(1) arrest induced by CCI-779 with ID(50) concentrations of <1 nM. In contrast, myeloma cells expressing wild-type PTEN were >1000-fold more resistant. Acute expression of a constitutively active AKT gene in CCI-779-resistant myeloma cells containing wild-type PTEN ...

Regulatory Mechanisms in Breast Cancer, 1991

Smad proteins transduce signals carried by the transforming growth factor (TGF-) cytokine superfa... more Smad proteins transduce signals carried by the transforming growth factor (TGF-) cytokine superfamily from receptor serine/threonine kinases at the cell surface to the nucleus, thereby affecting cell prolifera- tion, differentiation, as well as pattern formation during early vertebrate development. Smad4/DPC4, located at chromosome 18q21, was identified as a candidate tumor suppressor gene that is inactivated in nearly half of all

Cancer research, 2002

Recent work identifies the AKT kinase as a potential mediator of tumor expansionin multiple myelo... more Recent work identifies the AKT kinase as a potential mediator of tumor expansionin multiple myeloma. The finding of PTEN mutations in several myeloma cell lines suggests that loss of PTEN function may be one mechanism by which AKT activity is increased in ...

Oncogene, 1997

We have constructed a panel of substitution mutants which affect one or more of the putative cdk ... more We have constructed a panel of substitution mutants which affect one or more of the putative cdk target sites of the RB protein. We have examined the activity of these mutants relative to wild-type RB by both a transcriptional repression assay and by measuring growth suppression in vitro. We find that some phosphorylation site mutants of pRB can repress E2 transcription more strongly than wild-type RB. These mutants are partially resistant to phosphorylation by cdks and can arrest tumor cells in G1 in vitro. Our results indicate a functional correlation between the ability to repress E2F-dependent transcription and the ability to suppress tumor cell growth in vitro. In addition, we describe two classes of RB mutants: N-terminal truncated p56RB and a novel mutant of RB containing multiple substitutions near its nuclear localization signal. Both classes of RB mutants have greater activity than the wild-type protein. Because RB is a key regulator of cell cycle progression, expression o...

International Journal of Cancer, 2003

ELAC2 is a novel candidate cancer susceptibility gene located on chromosome 17p: Carriers of muta... more ELAC2 is a novel candidate cancer susceptibility gene located on chromosome 17p: Carriers of mutations in ELAC2 display a higher risk of developing prostate cancer. Overexpression of ELAC2 in tumor cells causes a delay in G2-M progression characterized by accumulation of cyclin B levels. Consistent with a function in mitosis, further biochemical analysis revealed that ELAC2 physically interacts with the gamma-tubulin complex. This is the first biologic insight into the function of this new putative cancer susceptibility gene, providing clues of how perturbation of ELAC2 might promote tumorigenesis through irregular cell division.

Prostate Cancer: Biology, Genetics, and the New …, 2001

Page 85. 5 Tumor Suppressor Genes in Prostate Cancer Robert Bookstein, MD 1. DEFINITION AND GENER... more Page 85. 5 Tumor Suppressor Genes in Prostate Cancer Robert Bookstein, MD 1. DEFINITION AND GENERAL PROPERTIES OF TUMOR SUPPRESSOR GENES The concept of tumor suppressor genes (TSGs) was originally ...

Cancer Research, May 15, 1999

Genetic alterations in the MMAC1 tumor suppressor gene (also referred to as PTEN or TEP1) occur i... more Genetic alterations in the MMAC1 tumor suppressor gene (also referred to as PTEN or TEP1) occur in several types of human cancers including glioblastoma. Growth suppression induced by overexpression of MMAC1 in cells with mutant MMAC1 alleles is thought to be mediated by the inhibition of signaling through the phosphatidylinositol 3-kinase pathway. However, the exact biochemical mechanisms by which MMAC1 exerts its growth-inhibitory effects are still unknown. Here we report that recombinant adenovirus-mediated overexpression of MMAC1 in three different MMAC1-mutant glioblastoma cell lines blocked progression from G0/G1 to S phase of the cell cycle. Cell cycle arrest correlated with the recruitment of the cyclin-dependent kinase (CDK) inhibitor, p27Kip1, to cyclin E immunocomplexes, which resulted in a reduction in CDK2 kinase activities and a decrease in levels of endogenous phosphorylated retinoblastoma protein. CDK4 kinase activities were unaffected, as were the levels of the CDK ...

The MMAC/PTEN tumor suppressor gene encodes for a phosphatase that recently has been shown to hav... more 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 ac tivity, without significantly altering Akt expression. The effects of func tional 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 phospho rylation of ...

Cancer research, 2002

Recent work identifies the AKT kinase as a potential mediator of tumor expansion in multiple myel... more Recent work identifies the AKT kinase as a potential mediator of tumor expansion in multiple myeloma. The finding of PTEN mutations in several myeloma cell lines suggests that loss of PTEN function may be one mechanism by which AKT activity is increased in this disease. Because PTEN-deficient myeloma cells may have up-regulated activity of the mammalian target of rapamycin (mTOR), downstream of AKT, they may be particularly sensitive to mTOR inhibition. To test this hypothesis, we challenged myeloma cell lines with CCI-779, a newly developed analogue of rapamycin and an efficient inhibitor of mTOR. Three of four PTEN-deficient cell lines with constitutively active AKT were remarkably sensitive to cytoreduction and G(1) arrest induced by CCI-779 with ID(50) concentrations of <1 nM. In contrast, myeloma cells expressing wild-type PTEN were >1000-fold more resistant. Acute expression of a constitutively active AKT gene in CCI-779-resistant myeloma cells containing wild-type PTEN ...

A candidate tumor suppressor gene, MMACJ/PTEN,located in human chromosome band 10q23, was recenti... more A candidate tumor suppressor gene, MMACJ/PTEN,located in human chromosome band 10q23, was recentiy identified based on sequence al terations 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 sped mens and cancer cell lines of many types for 10q23 allelic losses and MMACJ 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 lOq allelic loss reported for many cancers. Of 124 tumor spedmens exhibiting LOH that have been screened for MMAC1 alterations to date, we have detected variants in 13 (—10%)of these primary tumors; the highest frequency of variants was found in glioblastoma specimens (—23%). Novel alterations identified in this gene include a missense variant In a melanoma sample and a splicing variant and a...

Oncology and Therapy

Introduction: Next-generation sequencing has emerged as a clinical tool for the identification of... more Introduction: Next-generation sequencing has emerged as a clinical tool for the identification of actionable mutations to triage advanced colorectal cancer patients for targeted therapies. The literature is conflicted as to whether primaries or their metastases should be selected for sequencing. Some authors suggest that either site may be sequenced, whereas others recommend sequencing the primary, the metastasis, or even both tumors. Here, we address this issue head on with a meta-analysis and provide for the first time a set of sensible recommendations to make this determination. Methods: From our own series, we include 43 tumors from 13 patients including 14 primaries, 10 regional lymph node metastases, 17 distant metastases, and two anastomotic recurrences sequenced using the 50 gene Ion AmpliSeq cancer NGS panel v2. Results: Based on our new cohort and a metaanalysis, we found that * 77% of patient-matched primary-metastatic pairs have identical alterations in these 50 cancer-associated genes. Conclusions: Low tumor cellularity, tumor heterogeneity, clonal evolution, treatment status, sample quality, and/or size of the sequencing panel accounted for a proportion of the differential detection of mutations at primary and metastatic sites. The therapeutic implications of the most frequently discordant alterations (TP53, APC, PIK3CA, and SMAD4) are discussed. Our meta-analysis indicates that a subset of patients who fail initial therapy may benefit from sequencing of additional sites to identify new actionable genomic abnormalities not present in the initial analysis. Evidencebased recommendations are proposed.

Cancer Research, Jun 1, 1998

Mutated in multiple advanced cancers 1/phosphatase and tensin homo logue (MMACl/PTEN) is a novel ... more Mutated in multiple advanced cancers 1/phosphatase and tensin homo logue (MMACl/PTEN) is a novel tumor suppressor gene candidate located on chromosome 10 that is commonly mutated in human glioblastoma multiforme and several other cancer types. To evaluate the function of this gene as a tumor suppressor, we constructed a replication-defective adenovirus (MMCB) for efficient, transient transduction of MMAC1 into tumor cells. Infection of MMACl-mutated U87MG glioblastoma cells with MMCB resulted in dose-dependent exogenous MMAC1 protein expres sion as detected by Western blotting of cell lysates. In vitro proliferation of U87MG cells was inhibited by MMCB in comparison to several control adenoviruses at equal viral doses, implying a specific effect of MMAC1 expression. Anchorage-independent growth in soft agar was also inhibited by MMCB compared to control adenovirus. Tumorigenicity in nude mice of transiently transduced mass cell cultures was then assessed. MMCBinfected U87MG cells were almost completely nontumorigenic compared to untreated and several control adenovirus-treated cells at equal viral doses. These data support an in vivo tumor suppression activity of MMACl/PTEN and suggest that in vivo gene transfer with this recombi nant adenoviral vector has a potential use in cancer gene therapy.

Cancer Research, May 1, 2003

Approximately 30-40% of malignant glial tumors exhibit mutations in the tumor suppressor gene, PT... more Approximately 30-40% of malignant glial tumors exhibit mutations in the tumor suppressor gene, PTEN/MMAC. Additionally, these tumors are associated with (a) mutations in epidermal growth factor receptor (EGFR), leading to a pro-oncogenic constitutive activation, as well as amplification of its gene, and/or (b) mutations in p53, disrupting normal cellular homeostatic processes. Whereas PTEN/MMAC has been shown to possess antiangiogenic action, constitutively active EGFR or p53 gene defects have been associated with proangiogenic action. In this article, we asked if PTEN/MMAC gene transfer into human glioma cells that possess inactivating mutations of the PTEN/MMAC gene but also express either constitutively active EGFR (U87⌬EGFR cells) or possess an inactivating mutation of p53 (U251 cells) still display inhibited angiogenesis in orthotopic and ectopic models of gliomas. Human glioma xenografts treated with PTEN/MMAC gene transfer exhibited significantly decreased vascularity both in an orthotopic and in an ectopic model. Taken in combination, these results provide strong evidence of PTEN/ MMAC's role in regulating glioma angiogenesis even in the presence of strong proangiogenic signals provided by constitutive EGFR activation or p53 inactivation.

Cancer Research, May 15, 1999

Genetic alterations in the MMAC1 tumor suppressor gene (also referred to as PTEN or TEP1) occur i... more Genetic alterations in the MMAC1 tumor suppressor gene (also referred to as PTEN or TEP1) occur in several types of human cancers including glioblastoma. Growth suppression induced by overexpression of MMAC1 in cells with mutant MMAC1 alleles is thought to be mediated by the inhibition of signaling through the phosphatidylinositol 3-kinase pathway. However, the exact biochemical mechanisms by which MMAC1 exerts its growth-inhibitory effects are still unknown. Here we report that recombinant adenovirus-mediated overexpression of MMAC1 in three different MMAC1-mutant glioblastoma cell lines blocked progression from G 0 /G 1 to S phase of the cell cycle. Cell cycle arrest correlated with the recruitment of the cyclin-dependent kinase (CDK) inhibitor, p27 Kip1 , to cyclin E immunocomplexes, which resulted in a reduction in CDK2 kinase activities and a decrease in levels of endogenous phosphorylated retinoblastoma protein. CDK4 kinase activities were unaffected, as were the levels of the CDK inhibitor p21 Cip1 present in cyclin E immunocomplexes. Therefore, overexpression of MMAC1 via adenovirus-mediated gene transfer suppresses tumor cell growth through cell cycle inhibitory mechanisms, and as such, represents a potential therapeutic approach to treating glioblastomas.

Cancer Research, Nov 1, 2002

Smad proteins transduce signals carried by the transforming growth factor ␤ (TGF-␤) cytokine supe... more Smad proteins transduce signals carried by the transforming growth factor ␤ (TGF-␤) cytokine superfamily from receptor serine/threonine kinases at the cell surface to the nucleus, thereby affecting cell proliferation, differentiation, as well as pattern formation during early vertebrate development. Smad4/DPC4, located at chromosome 18q21, was identified as a candidate tumor suppressor gene that is inactivated in nearly half of all pancreatic carcinomas. For functional characterization of Smad4, a recombinant adenovirus encoding Smad4 (Ad-Smad4) was generated. When Smad4 was expressed in Smad4-null breast carcinoma cell line MDA-MB-468 using the recombinant adenovirus, TGF-␤ signaling was restored as determined by TGF-␤-dependent activity of plasminogen activator inhibitor 1 promoter and p21 expression. Infection with Ad-Smad4 in the presence of TGF-␤1 also resulted in an altered cell morphology that coincided with enhanced ␤1 integrin expression and reduced efficiency of colony formation in soft agar. In agreement with increased p21 expression, Smad4-expressing cells showed modest reduction in S phase. However, Smad4 expression did not lead to induction of apoptosis under normal culture conditions. Interestingly, when Smad4-expressing cells were detached and incubated in suspension, they underwent rapid apoptosis in a TGF-␤-dependent manner. Induction of apoptosis caused by loss of anchorage is known as anoikis. Anoikis is believed to prevent colonization elsewhere of detached cells. Additional characterization revealed an increase in the level of focal adhesion kinase 2 (or Pyk2) and activation of caspases 2, 3, 6, and 8 during anoikis because of Smad4 expression and restoration of TGF-␤ signaling. Because resistance to anoikis in tumor cells is thought to contribute to metastasis, our data suggest a functional basis for the strong correlation between defects in Smad4 and development of malignancy.

Journal of Cellular Biochemistry Supplement, Feb 1, 1994

Mutations of tumor suppressor genes are critical genetic alterations occurring during the genesis... more Mutations of tumor suppressor genes are critical genetic alterations occurring during the genesis and progression of human cancer, and consequently are candidates for use as surrogate endpoint biomarkers. The two most intensively studied suppressor genes, retinoblastoma (Rb) and p53, are mutated in approximately 20-50% of advanced-stage prostate cancers, but only rarely in early tumors. The precise DNA base changes, especially those affecting p53, may yield clues to relevant carcinogenic mechanisms. Increased expression of p53 in neoplastic cells, as detected by immunohistochemistry, may indicate mutation or a physiological response to DNA damage. Allelic losses of chromosome arms 8p and 16q are relatively common even in early prostate cancers. Quantitative measurement of allelic imbalance can be performed in preneoplastic or small neoplastic lesions, albeit with some technical challenge. The significance of whole-genome or regional allelic imbalance at various stages of prostatic oncogenesis has not been established.

Cancer Research

Recent work identifies the AKT kinase as a potential mediator of tumor expansion in multiple myel... more Recent work identifies the AKT kinase as a potential mediator of tumor expansion in multiple myeloma. The finding of PTEN mutations in several myeloma cell lines suggests that loss of PTEN function may be one mechanism by which AKT activity is increased in this disease. Because PTEN-deficient myeloma cells may have up-regulated activity of the mammalian target of rapamycin (mTOR), downstream of AKT, they may be particularly sensitive to mTOR inhibition. To test this hypothesis, we challenged myeloma cell lines with CCI-779, a newly developed analogue of rapamycin and an efficient inhibitor of mTOR. Three of four PTEN-deficient cell lines with constitutively active AKT were remarkably sensitive to cytoreduction and G(1) arrest induced by CCI-779 with ID(50) concentrations of <1 nM. In contrast, myeloma cells expressing wild-type PTEN were >1000-fold more resistant. Acute expression of a constitutively active AKT gene in CCI-779-resistant myeloma cells containing wild-type PTEN ...

Regulatory Mechanisms in Breast Cancer, 1991

Smad proteins transduce signals carried by the transforming growth factor (TGF-) cytokine superfa... more Smad proteins transduce signals carried by the transforming growth factor (TGF-) cytokine superfamily from receptor serine/threonine kinases at the cell surface to the nucleus, thereby affecting cell prolifera- tion, differentiation, as well as pattern formation during early vertebrate development. Smad4/DPC4, located at chromosome 18q21, was identified as a candidate tumor suppressor gene that is inactivated in nearly half of all

Cancer research, 2002

Recent work identifies the AKT kinase as a potential mediator of tumor expansionin multiple myelo... more Recent work identifies the AKT kinase as a potential mediator of tumor expansionin multiple myeloma. The finding of PTEN mutations in several myeloma cell lines suggests that loss of PTEN function may be one mechanism by which AKT activity is increased in ...

Oncogene, 1997

We have constructed a panel of substitution mutants which affect one or more of the putative cdk ... more We have constructed a panel of substitution mutants which affect one or more of the putative cdk target sites of the RB protein. We have examined the activity of these mutants relative to wild-type RB by both a transcriptional repression assay and by measuring growth suppression in vitro. We find that some phosphorylation site mutants of pRB can repress E2 transcription more strongly than wild-type RB. These mutants are partially resistant to phosphorylation by cdks and can arrest tumor cells in G1 in vitro. Our results indicate a functional correlation between the ability to repress E2F-dependent transcription and the ability to suppress tumor cell growth in vitro. In addition, we describe two classes of RB mutants: N-terminal truncated p56RB and a novel mutant of RB containing multiple substitutions near its nuclear localization signal. Both classes of RB mutants have greater activity than the wild-type protein. Because RB is a key regulator of cell cycle progression, expression o...

International Journal of Cancer, 2003

ELAC2 is a novel candidate cancer susceptibility gene located on chromosome 17p: Carriers of muta... more ELAC2 is a novel candidate cancer susceptibility gene located on chromosome 17p: Carriers of mutations in ELAC2 display a higher risk of developing prostate cancer. Overexpression of ELAC2 in tumor cells causes a delay in G2-M progression characterized by accumulation of cyclin B levels. Consistent with a function in mitosis, further biochemical analysis revealed that ELAC2 physically interacts with the gamma-tubulin complex. This is the first biologic insight into the function of this new putative cancer susceptibility gene, providing clues of how perturbation of ELAC2 might promote tumorigenesis through irregular cell division.

Prostate Cancer: Biology, Genetics, and the New …, 2001

Page 85. 5 Tumor Suppressor Genes in Prostate Cancer Robert Bookstein, MD 1. DEFINITION AND GENER... more Page 85. 5 Tumor Suppressor Genes in Prostate Cancer Robert Bookstein, MD 1. DEFINITION AND GENERAL PROPERTIES OF TUMOR SUPPRESSOR GENES The concept of tumor suppressor genes (TSGs) was originally ...