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John I Risinger

Publications and source records attributed to John I Risinger.

16 recordsLinked to original sources
PubMed2006

Characterization of an antibody that can detect the Kai1/CD82 murine metastasis suppressor.

BACKGROUND: Kai1, also known as CD82, is a member of the tetraspanin family (TM4SF). The human homolog, KAI1, is an activation antigen of T-cells and is a metastasis suppressor for prostate and other cancers. Little is known about the mouse protein because of the lack of antibody reagents. METHODS: Peptide immunized rabbits were used to generate polyclonal antibody to Kai1. The antibody was analyzed using immunoblotting, flow cytometry, and immunohistochemistry. RESULTS: This antibody specifically recognizes murine Kai1 protein, crossreacts with rat Kai1 but not with human KAI1. The normal tissue distribution of this protein in mice is shown to be similar to that of the human homolog. Interestingly, mouse prostatic epithelium showed differential expression within the lobes. CONCLUSION: This antibody, the first described that can specifically detect murine Kai1/CD82, should be very useful in addressing the mechanism of action of Kai1 in metastatic suppression.

Amino Acid Sequence↗
PubMed2006

Prediction of lymph node metastasis in patients with endometrioid endometrial cancer using expression microarray.

PURPOSE: To characterize the gene expression profiles of endometrioid endometrial cancers associated with lymph node metastasis in an effort to identify genes associated with metastatic spread. EXPERIMENTAL DESIGN: Tumors from 41 patients with endometrioid endometrial cancer grossly confined to the uterine cavity were evaluated. Positive lymph nodes were noted in 12 of 41 patients. RNA was analyzed for gene expression using the Affymetrix HG133A and HG133B GeneChip set, representing 45,000 array features covering >28,000 UniGene clusters. Data analysis was done using multidimensional scaling, binary comparison, and hierarchical clustering. Gene expression for several differentially expressed genes was examined using quantitative PCR. RESULTS: Gene expression data was obtained from 30,964 genes that were detected in at least 5% of the cases. Supervised analysis of node-positive versus node-negative cases indicated that 450 genes were significantly differentially expressed between the two classes at P < 0.005, 81 of which were differentially expressed by at least 2-fold at P < 0.005. Overexpressed genes included two cell cycle checkpoint genes, CDC2 and MAD2L1, which have previously been described in association with lymph node metastasis in other cancer types. The ZIC2 zinc finger gene was overexpressed in endometrial cancers with positive nodes versus those with negative nodes. CONCLUSION: Gene expression profiling of the primary tumors in patients with endometrioid endometrial cancers seems promising for identifying genes associated with lymph node metastasis. Future studies should address whether the status of nodal metastasis can be determined from the expression profiles of preoperative tissue specimens.

Carcinoma, Endometrioid↗
PubMed2006

Gene expression analysis of tumor infiltrating lymphocyte markers in endometrial cancers indicates no significant increases in those cases with microsatellite instability.

Microsatellite instability (MSI) is seen in many cancers and is the result of either a germline or somatic defect in the DNA mismatch repair system. Microsatellite instability is common in endometrial cancers occurring in about 25% of cases with endometrioid histology. Tumor infiltrating lymphocytes (TIL) are more prominent in colorectal cancer cases with MSI. The presence of increased TIL is associated with increased survival in these colorectal cancers, and is suggested as one possible mechanism to explain the increased survival rates in colorectal cancer patients with MSI positive cancers. Some degree of evidence indicates that increased TIL is also predictive of increased survival in endometrial cancer. The relative levels and states of activation of TIL in endometrial cancers with and without MSI has not been explored. Our previous data indicates that global gene expression patterns from MSI and non-MSI endometrial cancers are distinct, however TIL markers were not over-represented on statistically relevant gene lists that distinguish these groups. We further examined these pre-existing microarray data by directly querying transcripts present in the T-cell gene ontology (GO) group. No significant differences were observed between MSI and microsatellite stable (MSS) groups. Finally we directly examined a set of T-cell marker transcripts previously utilized to define increased activated and cytotoxic TIL in MSI positive colorectal cancers. Whereas colorectal cancers with MSI have been previously demonstrated to contain higher ratios of CD8/CD3 message levels we observed no difference in endometrial cancers. In addition, levels of CD3 indicated no increases in TIL in MSI positive cases and 2 markers of activation, granzyme B and IL-2R were not different in MSI positive and negative cancers. These data indicate that significant differences in TIL derived transcripts do not occur between endometrioid endometrial cancers with and without microsatellite instability.

Biomarkers, Tumor↗
PubMed2005

Reciprocal binding of CTCF and BORIS to the NY-ESO-1 promoter coincides with derepression of this cancer-testis gene in lung cancer cells.

Regulatory sequences recognized by the unique pair of paralogous factors, CTCF and BORIS, have been implicated in epigenetic regulation of imprinting and X chromosome inactivation. Lung cancers exhibit genome-wide demethylation associated with derepression of a specific class of genes encoding cancer-testis (CT) antigens such as NY-ESO-1. CT genes are normally expressed in BORIS-positive male germ cells deficient in CTCF and meCpG contents, but are strictly silenced in somatic cells. The present study was undertaken to ascertain if aberrant activation of BORIS contributes to derepression of NY-ESO-1 during pulmonary carcinogenesis. Preliminary experiments indicated that NY-ESO-1 expression coincided with derepression of BORIS in cultured lung cancer cells. Quantitative reverse transcription-PCR analysis revealed robust, coincident induction of BORIS and NY-ESO-1 expression in lung cancer cells, but not normal human bronchial epithelial cells following 5-aza-2'-deoxycytidine (5-azadC), Depsipeptide FK228 (DP), or sequential 5-azadC/DP exposure under clinically relevant conditions. Bisulfite sequencing, methylation-specific PCR, and chromatin immunoprecipitation (ChIP) experiments showed that induction of BORIS coincided with direct modulation of chromatin structure within a CpG island in the 5'-flanking noncoding region of this gene. Cotransfection experiments using promoter-reporter constructs confirmed that BORIS modulates NY-ESO-1 expression in lung cancer cells. Gel shift and ChIP experiments revealed a novel CTCF/BORIS-binding site in the NY-ESO-1 promoter, which unlike such sites in the H19-imprinting control region and X chromosome, is insensitive to CpG methylation in vitro. In vivo occupancy of this site by CTCF was associated with silencing of the NY-ESO-1 promoter, whereas switching from CTCF to BORIS occupancy coincided with derepression of NY-ESO-1. Collectively, these data indicate that reciprocal binding of CTCF and BORIS to the NY-ESO-1 promoter mediates epigenetic regulation of this CT gene in lung cancer cells, and suggest that induction of BORIS may be a novel strategy to augment immunogenicity of pulmonary carcinomas.

Antigens, Neoplasm↗
PubMed2005

Conditional expression of the CTCF-paralogous transcriptional factor BORIS in normal cells results in demethylation and derepression of MAGE-A1 and reactivation of other cancer-testis genes.

Brother of the Regulator of Imprinted Sites (BORIS) is a mammalian CTCF paralog with the same central 11Zn fingers (11ZF) that mediate specific interactions with varying approximately 50-bp target sites. Regulated in vivo occupancy of such sites may yield structurally and functionally distinct CTCF/DNA complexes involved in various aspects of gene regulation, including epigenetic control of gene imprinting and X chromosome inactivation. The latter functions are mediated by meCpG-sensitive 11ZF binding. Because CTCF is normally present in all somatic cells, whereas BORIS is active only in CTCF- and 5-methylcytosine-deficient adult male germ cells, switching DNA occupancy from CTCF to BORIS was suggested to regulate site specificity and timing of epigenetic reprogramming. In addition to 11ZF-binding paternal imprinting control regions, cancer-testis gene promoters also undergo remethylation during CTCF/BORIS switching in germ cells. Only promoters of cancer testis genes are normally silenced in all somatic cells but activated during spermatogenesis when demethylated in BORIS-positive germ cells and are found aberrantly derepressed in various tumors. We show here that BORIS is also expressed in multiple cancers and is thus itself a cancer-testis gene and that conditional expression of BORIS in normal fibroblasts activates cancer-testis genes selectively. We tested if replacement of CTCF by BORIS on regulatory DNA occurs in vivo on activation of a prototype cancer-testis gene, MAGE-A1. Transition from a hypermethylated/silenced to a hypomethylated/activated status induced in normal cells by 5-aza-2'-deoxycytidine (5-azadC) was mimicked by conditional input of BORIS and is associated with complete switching from CTCF to BORIS occupancy at a single 11ZF target. This site manifested a novel type of CTCF/BORIS 11ZF binding insensitive to CpG methylation. Whereas 5-azadC induction of BORIS takes only few hours, derepression of MAGE-A1 occurred 1 to 2 days later, suggesting that BORIS mediates cancer-testis gene activation by 5-azadC. Indeed, infection of normal fibroblasts with anti-BORIS short hairpin RNA retroviruses before treatment with 5-azadC blocked reactivation of MAGE-A1. We suggest that BORIS is likely tethering epigenetic machinery to a novel class of CTCF/BORIS 11ZF target sequences that mediate induction of cancer-testis genes.

Animals↗
PubMed2005

The microcephaly ASPM gene is expressed in proliferating tissues and encodes for a mitotic spindle protein.

The most common cause of primary autosomal recessive microcephaly (MCPH) appears to be mutations in the ASPM gene which is involved in the regulation of neurogenesis. The predicted gene product contains two putative N-terminal calponin-homology (CH) domains and a block of putative calmodulin-binding IQ domains common in actin binding cytoskeletal and signaling proteins. Previous studies in mouse suggest that ASPM is preferentially expressed in the developing brain. Our analyses reveal that ASPM is widely expressed in fetal and adult tissues and upregulated in malignant cells. Several alternatively spliced variants encoding putative ASPM isoforms with different numbers of IQ motifs were identified. The major ASPM transcript contains 81 IQ domains, most of which are organized into a higher order repeat (HOR) structure. Another prominent spliced form contains an in-frame deletion of exon 18 and encodes 14 IQ domains not organized into a HOR. This variant is conserved in mouse. Other spliced variants lacking both CH domains and a part of the IQ motifs were also detected, suggesting the existence of isoforms with potentially different functions. To elucidate the biochemical function of human ASPM, we developed peptide specific antibodies to the N- and C-termini of ASPM. In a western analysis of proteins from cultured human and mouse cells, the antibodies detected bands with mobilities corresponding to the predicted ASPM isoforms. Immunostaining of cultured human cells with antibodies revealed that ASPM is localized in the spindle poles during mitosis. This finding suggests that MCPH is the consequence of an impairment in mitotic spindle regulation in cortical progenitors due to mutations in ASPM.

Adult↗
PubMed2005

Gene expression profiling of microsatellite unstable and microsatellite stable endometrial cancers indicates distinct pathways of aberrant signaling.

Microsatellite instability (MSI) is a molecular phenotype present in approximately 25% of endometrial cancers. We examined the global gene expression profiles of early-stage endometrioid endometrial cancers with and without the MSI phenotype to test the hypothesis that MSI phenotype may determine a unique molecular signature among otherwise similar cancers. Unsupervised principal component analysis of the expression data from these cases indicated two distinct groupings of cancers based on MSI phenotype. A relatively small number of array features (392) at high statistical value (P < 0.001) were identified that drive the instability signature in these cancers; 109 of these transcripts differed by at least 2-fold. These data identify distinct gene expression profiles for MSI and microsatellite stable (MSS) cancers, which suggest that cancers with MSI develop in part by different mechanisms from their similar stable counterparts. In particular, we found evidence that two members of the secreted frizzled related protein family (SFRP1 and SFRP4) were more frequently down-regulated in MSI cancers as compared with MSS cancers. Down-regulation was accompanied by promoter hypermethylation for SFRP1. SFRP1 was hypermethylated in 8 of 12 MSI cancers whereas only 3 of 16 MSS cancers were methylated. The WNT target fibroblast growth factor 18 was found to be up-regulated in MSI cancers. These data classify histologically similar endometrioid endometrial cancers into two distinct groupings with implications affecting therapy and prevention.

Carcinoma, Endometrioid↗
PubMed2005

Microarray analysis of endometrial carcinomas and mixed mullerian tumors reveals distinct gene expression profiles associated with different histologic types of uterine cancer.

Previous studies using cDNA microarray have indicated that distinct gene expression profiles characterize endometrioid and papillary serous carcinomas of the endometrium. Molecular studies have observed that mixed mullerian tumors, characterized by both carcinomatous and sarcomatous components, share features that are characteristic of endometrial carcinomas. The objective of this analysis was to more precisely define gene expression patterns that distinguish endometrioid and papillary serous histologies of endometrial carcinoma and mixed mullerian tumors of the uterus. One hundred nineteen pathologically confirmed uterine cancer samples were studied (66 endometrioid, 24 papillary serous, and 29 mixed mullerian tumors). Gene expressions were analyzed using the Affymetrix Human Genome Arrays U133A and U133B Genechip set. Unsupervised analysis revealed distinct global gene expression patterns of endometrioid, papillary serous, mixed mullerian tumors, and normal tissues as grossly separated clusters. Two-sample t tests comparing endometrioid and papillary serous, endometrioid and mixed mullerian tumor, and papillary serous and mixed mullerian tumor pairs identified 1,055, 5,212, and 1,208 differentially expressed genes at P < 0.001, respectively. These data revealed that distinct patterns of gene expression characterize various histologic types of uterine cancer. Gene expression profiles for select genes were confirmed using quantitative PCR. An understanding of the molecular heterogeneity of various histologic types of endometrial cancer has the potential to lead to better individualization of treatment in the future.

Cluster Analysis↗
PubMed2004

The SPANX gene family of cancer/testis-specific antigens: rapid evolution and amplification in African great apes and hominids.

Human sperm protein associated with the nucleus on the X chromosome (SPANX) genes comprise a gene family with five known members (SPANX-A1, -A2, -B, -C, and -D), encoding cancer/testis-specific antigens that are potential targets for cancer immunotherapy. These highly similar paralogous genes cluster on the X chromosome at Xq27. We isolated and sequenced primate genomic clones homologous to human SPANX. Analysis of these clones and search of the human genome sequence revealed an uncharacterized group of genes, SPANX-N, which are present in all primates as well as in mouse and rat. In humans, four SPANX-N genes comprise a series of tandem duplicates at Xq27; a fifth member of this subfamily is located at Xp11. Similarly to SPANX-A/D, human SPANX-N genes are expressed in normal testis and some melanoma cell lines; testis-specific expression of SPANX is also conserved in mouse. Analysis of the taxonomic distribution of the long and short forms of the intron indicates that SPANX-N is the ancestral form, from which the SPANX-A/D subfamily evolved in the common ancestor of the hominoid lineage. Strikingly, the coding sequences of the SPANX genes evolved much faster than the intron and the 5' untranslated region. There is a strong correlation between the rates of evolution of synonymous and nonsynonymous codon positions, both of which are accelerated 2-fold or more compared to the noncoding sequences. Thus, evolution of the SPANX family appears to have involved positive selection that affected not only the protein sequence but also the synonymous sites in the coding sequence.

Amino Acid Sequence↗
PubMed2003

Microarray analysis reveals distinct gene expression profiles among different histologic types of endometrial cancer.

Previous studies of oncogene and tumor suppressor gene alterations have suggested that differences exist in the molecular pathogenesis of the various histological types of endometrial cancer. To elucidate further the molecular events involved in endometrial carcinogenesis, we examined global expression patterns of 16 nonendometrioid cancers (13 serous papillary and 3 clear cell), 19 endometrioid cancers, and 7 age-matched normal endometria using cDNA microarrays. Unsupervised analysis of gene expression identified 191 genes that exhibited >2-fold differences (P < 0.001) between the histological groups. Many genes were similarly dysregulated in both nonendometrioid and endometrioid cancers relative to normal endometria. Gene expression differences in only 24 transcripts could distinguish serous from endometrioid cancers, the two most common subgroups. These data provide the basis for investigation of previously unrecognized novel pathways involved in the development of endometrial cancers.

Endometrial Neoplasms↗
PubMed2003

Promoter hypermethylation as an epigenetic component in Type I and Type II endometrial cancers.

Epigenetic mechanisms that result in aberrant gene expression are a prominent feature of many cancer types. One main epigenetic mechanism for gene silencing involves promoter hypermethylation. Type I and type II endometrial cancers exhibit differing clinical, histologic, and molecular genetic characteristics. We hypothesize that these differences also extend to epigenetic phenomena. Promoter methylation analysis of 11 genes in a panel of endometrial cancers supports this hypothesis. These initial data indicate that promoter hypermethylation events occur frequently in type 1 cancer and were not detected in type II cancers using this panel of loci. These data tend to support the hypothesis that type I and type II endometrial cancers will exhibit distinct patterns of gene silencing based on promoter hypermethylation events.

Cell Transformation, Neoplastic↗
PubMed2002

Splice variants but not mutations of DNA polymerase beta are common in bladder cancer.

DNA polymerase beta (POLbeta) is a highly conserved protein that functions in base excision repair. Loss of the POLbeta locus on chromosome 8p is a frequent event in bladder cancer, and loss of POLbeta function could hinder DNA repair leading to a mutator phenotype. Both point mutations and large intragenic deletions of POLbeta have been reported from analysis of various tumor cDNAs but not from genomic DNA. We noticed that the breakpoints of the presumed rearrangements were delineated by exon-exon junctions, which could instead be consistent with alternative splicing of POLbeta mRNA. We tested the hypothesis that the reported intragenic deletion were splice variants by screening genomic DNA of human bladder tumors, bladder cancer cell lines, and normal bladder tissues for mutations or deletions in exons 1-14, exon alpha, and the promoter region of POLbeta. We found no evidence of somatic mutations or deletions in our sample set, although two polymorphisms were identified. Examination of cDNA from a subset of the original sample set revealed that truncated forms of POLbeta were surprisingly common. Forty-eight of 89 (54%) sequenced cDNA clones had large deletions, each beginning and/or ending exactly at exon-exon junctions. Because these deletions occur at exon-exon junctions and are seen in cDNA but not genomic DNA, they are consistent with alternative mRNA splicing. We describe 12 different splicing events occurring in 18 different combinations. Loss of exon 2 was the most frequent, being found in 42 of 49 (86%) of the variant sequenced clones. The splice variants appear to be somewhat more common and variable in bladder cancer cell lines and tumor tissues but occur at a high frequency in normal bladder tissues as well. We examine alternative splicing in terms of the information content of splice donor and acceptor site sequences, and discuss possible explanations for the predominant splicing event, the loss of exon 2.

Alternative Splicing↗
PubMed2002

Cell proliferation and apoptosis in human uterine leiomyomas and myometria.

To determine the role of cell proliferation and apoptosis in uterine leiomyoma growth, we studied protein expression of two major regulatory proteins of apoptosis -- Bcl-2 (anti-apoptotic) and Bax (pro-apoptotic) -- and two endogenous markers of cell replication - proliferating cell nuclear antigen (PCNA) and Ki-67 - in tumors and matched myometrium from premenopausal women. Conventional mitotic indices also were determined, and all proliferation data were correlated to tumor size. In situ end-labeling of fragmented DNA and routine histology were used to assess apoptosis. Our results showed that the apoptosis-regulating proteins (Bcl-2 and Bax) were expressed in the cytoplasm of the leiomyoma and myometrial smooth muscle cells throughout the menstrual cycle. Bax expression differed from Bcl-2 in that it also was found in the cytoplasm of vascular smooth muscle cells of the myometria and tumors. Both tumors and myometrial samples expressed 26-kDa and 21-kDa proteins that reacted with antibodies directed towards Bcl-2 and Bax, respectively. Apoptosis was not a prominent feature of uterine leiomyomas or myometrium. PCNA- and Ki-67-labeling and mitotic counts were significantly ( P<0.05) higher in leiomyomas than in matched myometrial samples. Proliferative activity was variable for individual tumors of the same patient and independent of tumor size. Our results suggest that altered apoptosis by overexpression of Bcl-2 or by decreased expression of Bax does not appear to be a major factor in uterine leiomyoma growth. We conclude that increased cell proliferation is the most significant contributor to growth and that the proliferative state is autonomous for each tumor in a given patient and is independent of tumor size.

Apoptosis↗
PubMed2002

Immortalization of human uterine leiomyoma and myometrial cell lines after induction of telomerase activity: molecular and phenotypic characteristics.

In vitro model systems for studying uterine leiomyomas are limited in that human-derived leiomyoma cells grow poorly in culture compared with normal myometrial cells and begin to senesce early, at approximately passage 10 in our studies. To our knowledge, a good in vitro human-derived cell culturing system for leiomyomas does not exist. In an attempt to fill this void, we have immortalized a uterine leiomyoma cell line by inducing telomerase activity, which allows cells to bypass their normal programmed senescence. Telomerase activity was induced by infecting the target (uterine leiomyoma and normal myometrial) cells with a retroviral vector containing hTERT, the gene for the catalytic subunit of telomerase. Subsequent analysis by RT-PCR and the telomeric repeat amplification protocol assay confirmed expression of the inserted gene and induction of telomerase activity in leiomyoma and myometrial cells. Analysis of cells for estrogen receptor-alpha and progesterone receptor proteins by Western blotting showed no change in expression of these proteins between the immortalized and parental leiomyoma and myometrial cells. Both immortalized and parental myometrial and leiomyoma cells expressed the smooth muscle-specific cytoskeletal protein alpha-actin and were negative for mutant p53 protein as evidenced by immunocytochemical staining. The immortalized leiomyoma and myometrial cells showed no anchorage-independent growth, with the exception of a small subpopulation of immortalized leiomyoma cells at a higher passage that did form two to three small colonies (per 50,000 cells) in soft agar. None of the immortalized cells were tumorigenic in nude mice. In conclusion, our data show the successful insertion of the hTERT gene into leiomyoma and myometrial cells and the immortalization of these cell lines without phenotypic alteration from the parental cell types (up to 200 population doublings). These cells should help to advance research in understanding the molecular pathways involved in the conversion of a normal myometrial cell to a leiomyoma cell and the mechanisms responsible for the growth of uterine leiomyomas. Answers to these questions will undoubtedly lead to the development of more effective treatment and intervention regimens for clinical cases of uterine leiomyoma.

Actins↗