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Biomedical subjects

Mark A Nelson

Publications and source records attributed to Mark A Nelson.

At least 19 recordsLinked to original sources
PubMed2006

Increased gene copy number of the transcription factor E2F1 in malignant melanoma.

Translocations and unique chromosome break points in melanoma will aid in the identification of the genes that are important in the neoplastic process. We have previously shown a unique translocation in malignant melanoma cells der(12)t(12;20). The transcription factor E2F1 maps to 20q11. Increased expression of E2F has been associated with the autonomous growth of melanoma cells, however, the molecular basis has not yet been elucidated. To this end, we investigated E2F1 gene copy number and structure in human melanoma cell lines and metastatic melanoma cases. Fluorescent in situ hybridization (FISH) analysis using a specific E2F1 probe indicated increased E2F1 gene copies in melanoma cell lines compared to normal melanocytes. We also observed increased copies of the E2F1 gene in lymph node metastases of melanoma. In addition, Western blot analysis demonstrated increased E2F1 protein levels in 8 out of 9 melanoma cell lines relative to normal melanocytes. Inhibition of E2F1 expression with RNAi also reduced melanoma cell growth. Our results suggest that the release of E2F activity by elevated E2F1 gene copy numbers may play a functional role in melanoma growth.

Blotting, Western↗
PubMed2005

Death-signal-induced relocalization of cyclin-dependent kinase 11 to mitochondria.

Fas receptor-Fas ligand interaction appears to be important in carcinogenesis, tumour outgrowth and metastasis. Emerging evidence suggests that CDK11 (cyclin-dependent kinase 11) plays a role in apoptosis and melanoma development. Here, we show that CDK11p110 protein kinase was cleaved after induction of apoptosis by Fas. The N-terminal portion of CDK11p110, CDK11p60, was translocated from the nucleus to the mitochondria. The targeting of CDK11p60 to mitochondria occurred as early as 12 h after treatment. Overexpression of EGFP (enhanced green fluorescent protein)-tagged CDK11p60 could partially break down the mitochondrial membrane potential, induce cytochrome c release and promote apoptosis. Reduction of endogenous CDK11p110 protein levels with siRNA (small interfering RNA) resulted in the suppression of both cytochrome c release and apoptosis. In addition, subcellular fractionation studies of Fas-mediated apoptosis demonstrated that CDK11p60 was associated with the mitochondrial import motor, mitochondrial heat shock protein 70. Taken together, our data suggest that CDK11p60 can contribute to apoptosis by direct signalling at the mitochondria, thereby amplifying Fas-induced apoptosis in melanoma cells.

Apoptosis↗
PubMed2005

The cyclin-dependent kinase 11 interacts with NOT2.

The caspase-processed cyclin-dependent kinase 11 (formerly known as PITSLRE) is implicated in apoptotic signaling. However, the mechanism of apoptotic signal transduction through CDK11(p46) is still unclear. We used a yeast two-hybrid screening strategy and identified NOT2 as an interacting partner of caspase-processed C-terminal kinase domain of CDK11 (CDK11(p46)). We demonstrate that CDK11(p46) directly interacts with NOT2 in vitro and in human cells. The NOT domain in the C-terminal part of NOT2 is responsible for the association between CDK11(p46) and NOT2. Both NOT2 and CDK11(p46) predominantly co-localized in the nucleus. Furthermore, we show that overexpression of NOT2 reduces luciferase mRNA and induces apoptosis. However, NOT2 is not phosphorylated by CDK11(p46). These findings suggest that CDK11 may contribute to apoptosis by regulating the activity of NOT2 independent of its kinase activity.

Apoptosis↗
PubMed2005

The cyclin-dependent kinase 11 interacts with 14-3-3 proteins.

Cyclin-dependent kinase 11 isoforms (CDK11) are members of the p34(cdc2) superfamily. They have been shown to play a role in RNA processing and apoptosis. In the present study, we investigate whether CDK11 interacts with 14-3-3 proteins. Our study shows that the putative 14-3-3 binding site (113-RHRSHS-118) within the N-terminal domain of CDK11(p110) is functional. Endogenous CDK11(p110) binds directly to 14-3-3 proteins and phosphorylation of the serine 118 within the RHRSHS motif seems to be required for the binding. Besides, CDK11(p110) is capable of interacting with several different isoforms of 14-3-3 proteins both in vitro and in vivo. The interaction of 14-3-3 gamma with CDK11(p110) occurs throughout the entire cell cycle and reaches maximum at the G2/M phase. Interestingly, 14-3-3 gamma shows strong interaction with N-terminal portion of caspase-cleaved CDK11(p110) (CDK11(p60)) product at 48 h after Fas treatment, which correlates with the maximal cleavage level of CDK11(p110) and the maximum activation level of CDK11 kinase activity during apoptosis. Collectively, these results suggest that CDK11 kinases could be regulated by interaction with 14-3-3 proteins during cell cycle and apoptosis.

14-3-3 Proteins↗
PubMed2005

Selenomethionine regulates cyclooxygenase-2 (COX-2) expression through nuclear factor-kappa B (NF-kappaB) in colon cancer cells.

Previously, we showed that selenomethionine (Se-Met) inhibits growth of colon cancer cells via suppressing COX-2 expression at both mRNA and protein level. However, the molecular mechanism by which Se-Met suppresses COX-2 expression remains to be elucidated. To this end, we transiently transfected HCA-7 cells with different COX-2 promoter constructs followed by Se-Met treatment (90 microM) for 12 h. The results suggested the role of nuclear factor-kappa B (NF-kappaB) in transcriptional regulation of COX-2. We also observed complete inhibition of DNA binding activity of NF-kappaB in Se-Met (90 microM) treated HCA-7 cells as shown by electrophoretic mobility shift assay (EMSA). Supershift assays with anti-p65 antibody identified p65 subunit in the protein complex. We further demonstrate dose-dependent inhibition of nuclear translocation of NF-kappaB/p65 in Se-Met treated HCA-7 cells, which could explain the observed reduction in DNA binding of NF-kappaB/p65. These results suggest that Se-Met regulates COX-2 at transcriptional level by modulating the activity of NF-kappaB transcription factor.

Colorectal Neoplasms↗
PubMed2005

A specific role for the TFIID subunit TAF4 and RanBPM in neural progenitor differentiation.

TAF4 is crucial for the activity of many transcription factors, including CREB, RAR and CSL/RBP-Jkappa, but the role for TAF4 in neural development is unknown. Embryonic cortical neural stem cells (NSC) showed strong expression of TAF4 that decreased during neuronal but not glial differentiation. In a protein-protein interaction screen, we identified the intracellular signaling factor RanBPM as a co-factor of TAF4. RanBPM co-localized with TAF4 in a subset of mitotic progenitors in vivo and endogenous TAF4 and RanBPM could be co-immunoprecipitated from NSC extracts. Interestingly, co-transfections of TAF4 and RanBPM led to a significant increase in the number of primary neurite processes but no increase in total neurite length, whereas RanBPM and a TAF4 isoform lacking the RanBPM-interacting domain exerted no significant effect. Our results demonstrate that temporally high expression levels of two factors considered to be relatively general in function can influence very specific events in neuronal differentiation.

Adaptor Proteins, Signal Transducing↗
PubMed2005

New developments in the staging of melanoma.

As the incidence of melanoma increases, so does the search for new staging techniques that may provide important prognostic information and aid in the detection of early metastatic disease. The application of molecular techniques may provide powerful new tools in this search. This review summarizes recent findings obtained by means of conventional RT-PCR, cDNA arrays, and proteomics in the investigation of human melanoma. The molecular tools discussed in this review demonstrate how global transcript and protein analysis might contribute not only to the staging of melanoma, but may hold great promise in improving the diagnosis and treatment of this disease.

Biomarkers, Tumor↗
PubMed2005

Studies into the anticancer effects of selenomethionine against human colon cancer.

Colorectal cancer is the third most frequent fatal malignant neoplasm in the United States and is expected to cause significant morbidity and mortality. The recent recall of cyclooxygenase-2 inhibitors from clinical trials highlights the need to develop other agents for cancer chemoprevention trials. Intervention strategies with selenium compounds represent a viable option to reduce colon cancer. Here we discuss epidemiologic studies and ongoing clinical trials with selenium. In addition, we discuss preclinical mechanistic studies that provide insights into the biochemical and molecular bases for the anticancer effects of selenomethionine.

Animals↗
PubMed2004

Regulation of stability of cyclin-dependent kinase CDK11p110 and a caspase-processed form, CDK11p46, by Hsp90.

CDK11p110 (cyclin-dependent kinase 11p110, formerly known as PITSLRE) is a member of the CDK superfamily. It associates with cyclin L and is involved in the regulation of transcription and in premRNA splicing. During staurosporine-, Fas- and tumour necrosis factor a-induced apoptosis, CDK11p110, is cleaved by caspases to generate smaller 46-50 kDa proteins containing the catalytic kinase domain. Ectopic expression of the caspase-processed form CDK11p46 induces apoptosis. The mechanisms that regulate activation and stability of CDK11 isoforms are still unclear. In the present study, we demonstrate that in human melanoma cells CDK11p110 and CDK11p46 interact with Hsp90 (heat-shock protein 90) and its co-chaperone cdc37. Furthermore, we show that the treatment of cells with the Hsp90-specific inhibitor geldanamycin leads to ubiquitination and enhanced degradation of both CDK11p110 and CDK11p46 through a proteasome-dependent pathway. We also determined that geldanamycin-triggered degradation of CDK11p46 slows down the progression of apoptosis. These results indicate that Hsp90 and cdc37 stabilize CDK11 kinase, and suggest that this stabilization is crucial for its pro-apoptotic function.

Apoptosis↗
PubMed2004

Isolation and characterization of the human Cdc2L1 gene promoter.

CDK11 (cyclin-dependent kinase 11, formerly known as PITSLRE) is a member of the p34cdc2-related kinases. It has been previously shown to be involved in a variety of different cellular processes including RNA processing, apoptosis, and cell cycle progression. It is encoded by two different but highly similar genes, Cdc2L1 (cell division control 2 like 1) and Cdc2L2 (cell division control 2 like 2). Previous studies from our group identified and characterized the transcriptional regulation of the human Cdc2L2 gene promoter. The current studies identify and characterize the Cdc2L1 gene promoter. We cloned the promoter and elucidated the different transcriptional regulatory elements that reside within the 5' region of the gene. Deletion analysis of the promoter showed a region of nucleotides -152 to +11 to be necessary for basal transcription of the Cdc2L1 gene. Sequencing analysis found this region of the promoter to be highly GC-rich but is lacking both TATA and CAAT boxes. There are several different transcription factor binding sites that are consensus or near consensus found within this region. The potential binding sites include two Ets-1 sites, one Skn-1 site, and one E2F-1 site. Transfection studies of various site-directed mutagenesis clones for these different sites revealed that both Ets-1 sites play critical roles in sustained transcriptional activity as well as Skn-1. Chromatin immunoprecipitation of the endogenous promoter with Ets-1 and Skn-1 verified an in vivo association of Ets-1 and Skn-1 transcription factors with the endogenous promoter. These results, in addition to our Cdc2L2 results, lead to the further comprehension of the fundamental mechanisms dictating CDK11 gene expression through the Cdc2L1 gene promoter.

5' Flanking Region↗
PubMed2004

p27Kip1 and cyclin D1 are necessary for focal adhesion kinase regulation of cell cycle progression in glioblastoma cells propagated in vitro and in vivo in the scid mouse brain.

We have reported previously that the expression of focal adhesion kinase (FAK) is elevated in glioblastomas and that expression of FAK promotes the proliferation of glioblastoma cells propagated in either soft agar or in the C.B.17 severe combined immunodeficiency (scid) mouse brain. We therefore determined the effect of FAK on cell cycle progression in these cells. We found that overexpression of wild-type FAK promoted exit from G(1) in monolayer cultures of glioblastoma cells, enhanced the expression of cyclins D1 and E while reducing the expression of p27(Kip1) and p21(Waf1), and enhanced the kinase activity of the cyclin D1-cyclin-dependent kinase-4 (cdk4) complex. Transfection of the monolayers with a FAK molecule in which the autophosphorylation site is mutated (FAK397F) inhibited exit from G(1) and reduced the expression of cyclins D1 and E while enhancing the expression of p27(Kip1) and p21(Waf1). Small interfering RNA (siRNA)-mediated down-regulation of cyclin D1 inhibited the enhancement of cell cycle progression observed on expression of wild-type FAK, whereas siRNA-mediated down-regulation of cyclin E had no effect. siRNA-mediated down-regulation of p27(Kip1) overcame the inhibition of cell cycle progression observed on expression of FAK397F, whereas down-regulation of p21(Waf1) had no effect. These results were confirmed in vivo in the scid mouse brain xenograft model in which propagation of glioblastoma cells expressing FAK397F resulted in a 50% inhibition of tumor growth and inhibited exit from G(1). Taken together, our results indicate that FAK promotes proliferation of glioblastoma cells by enhancing exit from G(1) through a mechanism that involves cyclin D1 and p27(Kip1).

Animals↗
PubMed2004

Selenomethionine induces sustained ERK phosphorylation leading to cell-cycle arrest in human colon cancer cells.

Selenomethionine (SeMet) is being tested alone and in combination with other agents in cancer chemoprevention trials. However, the molecular targets and the signaling mechanism underlying the anticancer effect of this compound are not completely clear. Here, we provide evidence that SeMet can induce cell-growth arrest and that the growth inhibition is associated with S-G2/M cell-cycle arrest. Coincidentally with the cell-cycle arrest, we observed a striking increase in cyclin B as well as phosphorylation of the cyclin-dependent kinase Cdc2. Since activation of the mitogen-activated protein kinase (MAPK) cascade has been associated with cell-cycle arrest and growth inhibition, we evaluated the activation of extracellular signal-regulated kinase (ERK). We found that SeMet induced phosphorylation of the MAPK ERK in a dose-dependent manner. We also demonstrate phosphorylation of ribosomal S6 kinase (p90RSK) by SeMet. Additionally, we show phosphorylation of histone H3 in a concentration-dependent manner. Furthermore, the phosphorylation of p90RSK and histone H3 were both antagonized by the MEK inhibitor U0126, implying that SeMet-induced phosphorylation of p90RSK and histone H3 are at least in part ERK pathway dependent. Based on these results, we propose that SeMet induced growth arrest and phosphorylation of histone H3 are mediated by persistent ERK and p90RSK activation. These new data provide valuable insights into the biological effects of SeMet at clinically relevant concentrations.

Antineoplastic Agents↗
PubMed2004

Identification and characterization of the human Cdc2l2 gene promoter.

The CDK11 (cyclin-dependent kinase 11, formerly known as PITSLRE) protein kinases are part of the large family of p34(cdc2)-related kinases and have been shown to play a role in cell cycle progression, RNA processing and apoptosis. They are encoded by two genes-cell division control like 1 (Cdc2L1) and cell division control like 2 (Cdc2L2). To date, little is known about the transcription factors controlling their expression. To understand the mechanisms underlying the regulation of CDK11 gene expression, we cloned and identified the Cdc2L2 promoter and determined its transcriptional regulatory elements. By deletion analysis, a region between nucleotides -145 and +10 was identified to be critical for basal level transcription of the Cdc2L2 gene. Sequencing analysis revealed that the proximal promoter of the Cdc2L2 gene is GC rich and does not contain TATA and CAAT boxes. However, multiple consensus and near consensus transcription factor binding sites were found to be present in this region, such as two Ets-1, one cAMP-responsive element (CRE) and one TCF11/LCR-F1/Nrf1 binding sites. Site-directed mutagenesis and transfection studies revealed that all these binding sites were necessary to achieve sustained transcriptional activity. Electrophoretic mobility shift assay confirmed that transcription factors Ets-1 and CREB bind to the Cdc2L2 promoter elements, indicating their potential role in the transcriptional regulation of Cdc2L2 gene. More importantly, Ets-1, CREB and phosphorylated CREB were found binding to the endogenous Cdc2L2 promoter using chromatin immunoprecipitation (CHIP) assay. Our results provide the foundation for further studies into the regulation of Cdc2L2 gene expression in normal homeostasis and cancer.

5' Flanking Region↗
PubMed2004

Chemoprevention of prostate cancer with selenium: an update on current clinical trials and preclinical findings.

Prostate cancer is the most common cancer diagnosed and the second leading cause of cancer-related deaths in men in the United States. The etiological factors that give rise to prostate cancer are not known. Therefore, it is not possible to develop primary intervention strategies to remove the causative agents from the environment. However, secondary intervention strategies with selenium (Se) compounds and other agents represent a viable option to reduce the morbidity and mortality of prostate cancer. In this review, we discuss ongoing clinical trials. In addition, we discuss preclinical mechanistic studies that provide insights into the biochemical and molecular basis for the anti-carcinogenic activity of both inorganic and organic forms of Se.

Clinical Trials as Topic↗