D-type cyclins complex with the androgen receptor and inhibit its transcriptional transactivation ability.

D-type cyclins regulate distinct cellular processes, such as mitotic cell cycle control, differentiation, and transcription. We have previously shown that the D-type cyclins are critical for the androgen-dependent proliferation of prostate cells. Here, we sought to determine whether cyclin D1 directly influences the transactivation potential of the androgen receptor, a transcription factor that strongly influences androgen-dependent proliferation. We found that ligand-mediated transcriptional activation of a physiological target, prostate-specific antigen, by the androgen receptor was inhibited by cyclins D1 and D3. The ability of D-type cyclins to inhibit androgen receptor transactivation was not shared with other cyclins, and cyclin D1 was as effective as dominant negative mutants of the androgen receptor in inhibiting transactivation. This function of cyclin D1 was independent of its role in cell cycle progression and is likely elicited through its ability to form a specific complex with the androgen receptor. These data underscore the various mechanisms through which the androgen receptor is regulated and also point to a negative feedback role for cyclin D1 in controlling androgen-dependent growth.

Identification of a new MAGE gene with tumor-specific expression by representational difference analysis.

Human genes expressed exclusively in tumors and male germ line cells, such as those of the MAGE, BAGE, and GAGE families, encode antigens recognized by T lymphocytes, which are potentially useful for antitumor immunotherapy. To identify new genes of this type, we generated cDNA populations enriched in sequences expressed only in testis and melanoma, using the representational difference analysis approach. A testis cDNA library enriched by subtraction with cDNA from four other normal tissues was hybridized with radiolabeled melanoma cDNA enriched by subtraction with normal skin cDNA. A cDNA fragment sharing significant homology with MAGE genes was identified, and a cosmid containing this new gene, named MAGE-C1, was isolated. MAGE-C1 is composed of four exons and encodes a putative protein of 1142 amino acids. It is about 800 residues longer than the other MAGE proteins due to the insertion of a large number of short repetitive sequences in front of the MAGE-homologous sequence. The MAGE-C1 gene appears to be located on band Xq26, whereas the MAGE-A and MAGE-B genes are located on Xq28 and Xp21, respectively. Like other MAGE genes, MAGE-C1 is expressed in a significant proportion of tumors of various histological types, whereas it is silent in normal tissues except testis. It is probable, therefore, that like other MAGE genes, MAGE-C1 encodes antigens that may constitute useful targets for cancer immunotherapy because of their strict tumoral specificity.

Loss of heterozygosity for 10q22-10qter in malignant melanoma progression.

Karyotypic and molecular data indicate that genetic events involving the chromosome region 10q22-10qter may be related to tumorigenesis in malignant melanoma. To test this we analyzed 10 polymorphic microsatellite repeats in the region 10q22-qter, using a polymerase chain reaction-based assay for loss of heterozygosity and DNA isolated from normal and tumor tissue from 26 individuals with malignant melanoma. The samples included 19 paired normal and malignant tissues representing various stages of melanoma as well as 7 cases in which samples from at least 2 different points in time during tumor progression were available. Our findings indicate that loss of heterozygosity of 10q22-10qter is a frequent event, that the observed loss of heterozygosity does not result from whole chromosome loss, and that it is associated with tumor progression. Finally, the appearance of new alleles in two of the tumors may indicate the involvement of DNA replication errors in melanoma analogous to such events in other tumor types.

A defined region of loss of heterozygosity at 11q23 in cutaneous malignant melanoma.

Karyotypic and molecular data indicate that genetic alterations of the long arm of chromosome 11 (11q) may be involved in malignant melanoma. To test this we analyzed 5 polymorphic microsatellite repeats on 11q using a PCR-based assay for loss of heterozygosity in normal and tumor tissues from 24 individuals with cutaneous malignant melanoma of various stages. Our findings indicate that a tumor suppressor gene that plays a role in malignant melanoma is located on the long arm of chromosome 11, likely within a 51 cM region at 11q23. Its loss appears to be a late event in tumor progression and may serve as an indicator for a less favorable clinical outcome.

Identification of a human gene encoding a homologue of Saccharomyces cerevisiae EXO1, an exonuclease implicated in mismatch repair and recombination.

The EXO1 gene was identified in Saccharomyces cerevisiae as a gene encoding an exonuclease that interacts with MSH2 and functions in mismatch repair and genetic recombination. To understand the role of EXO1 in higher eukaryotes, we identified the human EXO1 gene. The hEXO1 predicted amino acid sequence shares 26.6% identity with the S. cerevisiae EXO1 amino acid sequence. The human and S. cerevisiae proteins showed a similar ability to complement the mutator phenotype of S. cerevisiae rad27 mutants indicating that the two proteins are functionally similar. There appear to be two forms of hEXO1 that differ by the COOH-terminal 1 and 44 amino acids, respectively, and these appear to result from alternative RNA splicing. The hEXO1 gene consists of 14 exons and is transcribed to yield a 3-kb mRNA. Radiation hybrid and fluorescence in situ hybridization mapping studies indicate that the human gene is located at 1q42.2-qter. Northern blot analysis demonstrates that hEXO1 is expressed in high levels in testis; elevated expression was also observed in thymus and colon and to a lesser extent in small intestine, placenta, spleen, and ovary.

Elevated cyclins and cyclin-dependent kinase activity in the rhabdomyosarcoma cell line RD.

An important early event in the differentiation of skeletal muscle cells is exit from the cell cycle, after which full expression of the muscle phenotype occurs. Rhabdomyosarcoma (RMS), a tumor of skeletal muscle origin, expresses a number of muscle-specific proteins, including MyoD; however, these cells fail to arrest or differentiate when cultured in differentiation medium (DM). To determine the basis for the failure of RMS cells to differentiate or arrest, we studied the molecular response of the embryonal RMS cell line, RD, to culture in DM. Under these conditions, the retinoblastoma protein (RB) was primarily in the hyperphosphorylated state. This is in contrast to myoblasts cultured in DM, in which the hypophosphorylated form of RB is exclusively present. Measurements of the expression and activities of cyclin-dependent kinases (cdks) cdk2 and cdk4 indicated that RD cells maintained higher levels than do myoblasts, and the activity and abundance of these proteins did not significantly decrease upon culture in DM in RD cells, as they did in myoblasts. Similarly, elevated expression of cyclins D1, E, and A was observed in RD cells. Interestingly, cdk inhibitors are expressed in RD cells, with p16ink4 expression markedly elevated relative to myoblasts. Ectopic expression of p21cip1, p16ink4, or p27kip1 caused a growth arrest of RD cells but not detectable expression of a myogenic marker. Furthermore, a constitutively active RB protein could also inhibit the growth of RD cells without inducing myogenic differentiation. Taken together, these data suggest that the elevated levels of cdk2 and/or cdk4 observed in RD cells contribute to the inability of RD cells to growth arrest when cultured in DM but that these activities alone are not responsible for the failure of RD cells to differentiate.

Characterization of the GAGE genes that are expressed in various human cancers and in normal testis.

The GAGE-1 gene was identified previously as a gene that codes for an antigenic peptide, YRPRPRRY, which was presented on a human melanoma by HLA-Cw6 molecules and recognized by a clone of CTLs derived from the patient bearing the tumor. By screening a cDNA library from this melanoma, we identified five additional, closely related genes named GAGE-2-6. We report here that further screening of this library led to the identification of two more genes, GAGE-7B and -8. GAGE-1, -2, and -8 code for peptide YRPRPRRY. Using another antitumor CTL clone isolated from the same melanoma patient, we identified antigenic peptide, YYWPRPRRY, which is encoded by GAGE-3, -4, -5, -6, and -7B and which is presented by HLA-A29 molecules. Genomic cloning of GAGE-7B showed that it is composed of five exons. Sequence alignment showed that an additional exon, which is present only in the mRNA of GAGE-1, has been disrupted in gene GAGE-7B by the insertion of a long interspersed repeated element retroposon. These GAGE genes are located in the p11.2-p11.4 region of chromosome X. They are not expressed in normal tissues, except in testis, but a large proportion of tumors of various histological origins express at least one of these genes. Treatment of normal and tumor cultured cells with a demethylating agent, azadeoxycytidine, resulted in the transcriptional activation of GAGE genes, suggesting that their expression in tumors results from a demethylation process.

Genetic instability leads to loss of both p53 alleles in a human glioblastoma.

Little is known about the relationship between genetic recombination mechanisms and loss of tumour suppressor genes in solid tumours. Here, we demonstrate deletion and truncation of both p53 alleles in a primary human glioblastoma and a derived cell line as the combined result of a t(17;20) reciprocal translocation and a 1.1 Mbp genomic deletion on chromosome 17p, starting in intron 4 of the p53 gene and ending at the telomeric CA-repeat marker D17S960. These results (i) suggest that genetic instability can lead to loss of tumour suppressor gene function in solid cancers, (ii) provide mapping of one such recombination event at the nucleotide level, and (iii) establish the orientation of the p53 gene on chromosome 17 as: centromere 5'-3'-telomere.

The retinoblastoma tumor suppressor inhibits cellular proliferation through two distinct mechanisms: inhibition of cell cycle progression and induction of cell death.

Studies aimed at examining the precise function(s) of the retinoblastoma tumor suppressor protein, RB, have been hindered by the rapid phosphorylation and inactivation of ectopically expressed RB which occurs in the majority of cell types. Therefore, ectopically expressed RB is a poor inhibitor of cellular proliferation. We have designed constitutively active RB proteins, PSM-RB, that cannot be inactivated by phosphorylation. Using these proteins, we show that unlike wild-type RB, PSM-RB proteins inhibit cell cycle progression in a broad range of tumor cell types. Furthermore, unlike p16ink4a, PSM-RB is also a potent inhibitor of cell cycle progression in RB-deficient tumor cells. Surprisingly, we identified a tumor cell line that is resistant to the cell cycle inhibitory effects of PSM-RB. This finding challenges the hypothesis that RB must be inactivated in all cells for cell cycle progression to occur. Further characterization of this 'resistant' tumor line revealed that proliferation of these cells is still inhibited by PSM-RB. We show that this is due to PSM-RB-induced cell death. As such, these studies are the first to show that RB inhibits cellular proliferation through at least two distinct mechanisms - inhibition of cell cycle progression and induction of cell death.

Allelic deletion mapping on chromosome 5 in human carcinomas.

We analysed allelic deletions on chromosome 5 in microdissected human non-small cell lung cancers. Thirty-four primary squamous cell carcinomas, 15 primary adenocarcinomas and five regional lymph node metastases were investigated for loss of heterozygosity (LOH) in chromosomal region 5p15-q21. The sites analysed included the APC tumor suppressor gene at 5q21, five polymorphic microsatellite markers and the putative tumor suppressor locus del-27, that was assigned to chromosomal region 5p13-12 by fluorescence in situ hybridization (FISH) analysis. Allelic deletions encompassed larger genomic regions more often in squamous cell carcinomas than in adenocarcinomas. The del-27 amd APC regions were identified as two distinct regions with the highest LOH frequencies within 5p15-q21. In squamous cell carcinomas LOH frequencies were 73% at the del-27 and 70% at the APC locus. In adenocarcinomas LOH at the del-27 and APC loci occurred in 38% of the informative cases. Allelic deletion of the APC gene and at the del-27 locus was also detected in the metastases. The results suggest involvement of at least two tumor suppressor genes on chromosome 5 in lung tumorigenesis.

Isolation of DICE1: a gene frequently affected by LOH and downregulated in lung carcinomas.

In the development and progression of sporadic tumors multiple tumor suppressor genes are inactivated that may be distinct from predisposing cancer genes. Previously, a tumor suppressor locus on human chromosome 13q14 that is distinct from the retinoblastoma predisposing gene 1 (RB1) has been identified in lung, head and neck, breast, ovarian and prostate tumors. By an approach that combines genomic difference cloning and positional cloning we isolated the cDNA of a novel gene (DICE1) located at 13q14.12-14.2. The DICE1 gene is highly conserved in evolution and its mRNA is expressed in a wide variety of fetal and adult tissues. The DICE1 cDNA encodes a predicted protein of 887 amino acids corresponding to an 100 kD protein that shows 92.9% identity to the carboxy-terminal half of the mouse EGF repeat transmembrane protein DBI-1. The DBI-1 protein interferes with the mitogenic response to insulin-like growth factor 1 (IGF-I) and is presumably involved in anchorage-dependent growth. When compared to normal lung tissue expression of the DICE1 mRNA was reduced or undetectable in the majority of non-small cell lung carcinomas analysed. The location of the DICE1 gene in the region of allelic loss, its high evolutionary conservation and the downregulation of expression in carcinoma cells suggests that DICE1 is a candidate tumor suppressor gene in non-small cell lung carcinomas and possibly in other sporadic carcinomas.

Isoforms of the human PDZ-73 protein exhibit differential tissue expression.

Patients with renal and colon cancer frequently develop IgG autoantibodies toward the NY-CO-38/PDZ-73 antigen, a protein of 652 amino acids (73 kDa) which contains three copies of the PDZ protein-protein interaction domain. The gene encoding PDZ-73 mapped to chromosome 11p15.4-p15.1. Additional tissue-specific isoforms were identified: PDZ-45, which lacks the third PDZ domain and the putative PEST protein degradation motif, is expressed in kidney, colon, small intestine, brain and testis; PDZ-54 and PDZ-59, which also lack the third PDZ domains, have unique carboxyl terminal amino acids and are expressed in brain, kidney, bladder, colon cancer and renal cancer; and a putative PDZ-37 isoform, containing only the third PDZ domain, that is expressed in the central nervous system. Immunohistochemical staining with anti-PDZ 73 monoclonal antibodies showed strong cytoplasmic reactivity in epithelial cells of the small intestine, colon and kidney tubules, with a prominent apical staining pattern in cells of the small intestine. The reactivity pattern of the antibodies with various tissues correlated with the mRNA expression pattern of the PDZ-45 isoform. The existence of multiple PDZ-73 isoforms with variations in tissue distribution, PDZ domains, protein degradation sequences and carboxyl terminal structure indicate that these isoforms have distinct tissue-specific functions.

Differential silver-positive nucleolus organizer region activity in normal and malignant murine tissues.

Transcriptionally active nucleolus organizer regions (NORs) were analyzed using a silver-staining technique. The levels of silver-positive NOR activity for normal murine bone marrow and thymus cells were established, and significant differences in ribosomal RNA gene activity were observed. When tumor cells originating from these two tissue types were also studied, significant differences were seen not only between the normal and malignant tissues, but between the two tumor types as well. These differences in ribosomal RNA gene activity with respect to cell type and malignancy may be useful diagnostically.

Mutation and expression of TP53 in malignant melanomas.

Mutations of the TP53 gene are the most common genetic alterations in human malignancies. Overexpression of the p53 protein has been reported in high frequencies in all types of skin cancer. To determine the role of TP53 in the pathogenesis of malignant melanoma, we investigated the expression of p53 in 12 cell lines and 145 primary and metastatic lesions by immunohistochemistry. Overexpression of p53 was predominantly detected in the cytoplasm of the cells in 96 (66%) tumor and 12 (93%) cell lines. In contrast to findings in other tumor types, in melanomas immunoreactive cells were found in clusters or as scattered single cells. In primary melanomas, the frequency of p53 overexpression did not correlate with tumor thickness. Nucleotide sequencing of TP53 genes of 24 melanoma tumors/cell lines demonstrated point mutations in seven samples, all coding for mutant p53 protein species. The frequency of TP53 alterations of 20%-30% is lower than in other skin tumor types. Notably, immunohistochemistry was not a suitable method to distinguish overexpression of wild-type p53 from mutant species, since cell lines/tumors with TP53 mutations did not show distinctive staining patterns. The mutation pattern in six out of seven lesions was similar to that caused by ultraviolet light damage. This finding may be regarded a further indication for a pathogenetic role of UV light damage in at least a subgroup of malignant melanomas.

Cytogenetic characterization and DNA content analysis of Ad 2-transformed rat embryo brain cells.

An adenovirus type 2-transformed rat embryo brain cell line (F 17 A2AS) and two of its subclones (F 17 CI, F 17 C2) have been studied using chromosome banding and flow cytometric techniques. In determining the modal chromosome number, a bimodal distribution was observed in each of the cell lines. In each case the first numerical mode fell in the hypertriploid or hypotetraploid range while the second represented a mode in the hypertetraploid range. Analysis of Giemsa-banded karyotypes revealed the presence of several marker chromosomes. In most cases the origins of the markers could be established from their characteristics Giemsa-banding patterns. In particular, chromosome 2 was involved in the formation of several marker chromosomes. The DNA content analysis confirmed the bimodal chromosome distribution with values in the near tetraploid and near pentaploid range for each cell line.

[Various activities of the nucleolus organizer region in normal and leukemic bone marrow cells: semiquantitative data and computer- assisted image analysis by silver staining]. / Razlichnaia aktivnost' iadrushkovykh organizatorov v normal'nom i leikoznom kostnom mozge: polukolichestvennye dannye i otsenka okraski serebrom s pomoshch'iu komp'iuternogo analiza izobrazheniia.

A cytochemical technique with silver nitrate staining was used to study the nucleolar organizer activity in metaphase spread of bone marrow cells from 13 patients with acute lymphocytic leukemia (ALL), 11 patients with chronic myelogenous leukemia (CML), 7 patients with acute myelogenous leukemia (AML), and 4 normal persons. Additionally, computer-assisted image analysis was used to quantitate the amount of silver staining in interphase nuclei of bone marrow cells from 6 untreated ALL patients, 3 normal subjects and 1 bone-marrow-transplant recipient. The results obtained have indicated that the nucleolar organizer reactivity (NOR) is significantly lower in the control group than that in ALL patients. NOR activity level is significantly lower in both CML patients in chronic phase, and AML patients than in the ALL group, and is similar to that in the control group. When the data obtained for the interphase nuclei were compared with those obtained for the metaphase spread, a strong correlation was recorded between the fraction of bone-marrow metaphases stained positively with silver, the average number of silver-positive nucleolar organizer regions per metaphase, and the amount of silver staining in the interphase nuclei. Silver staining used for the detection of these disease-related differences in NOR activity can serve as a diagnostic procedure in evaluating human leukemias. The computer-assisted image analysis of bone marrow cell interphase nuclei would be useful for more accurate resolving biological and medical problems.

FOXO animal models reveal a variety of diverse roles for FOXO transcription factors.

The Foxo subfamily of FOX transcription factors plays a variety of roles in a broad assortment of diverse physiological processes including cellular differentiation, tumor suppression, metabolism, cell cycle arrest, cell death and protection from stress. Animal models have proved to be invaluable tools in furthering our understanding of the role of particular genes in complex organismal processes. Multiple animal models in diverse species, including Caenorhabditis elegans, Drosophila. melanogaster and the laboratory mouse, exist for the Foxo family of transcription factors. Foxo genes are highly conserved throughout the evolution and each of these model systems has provided valuable insight into the roles of Foxo factors. Many roles are conserved among the different model organisms. Several Foxo-related animal model systems are reviewed here along with the knowledge gleaned to date from each model system.

cDNA and protein sequence, genomic organization, and analysis of cis regulatory elements of mouse and human PLVAP genes.

PV-1 is a novel protein component of the endothelial fenestral and stomatal diaphragms. PV-1 cDNA and protein sequences are highly conserved across species. The conserved extracellular domain features found in rat, mouse, and human PV-1 protein are four N-glycosylation sites, two coiled-coil domains, a proline-rich region, and even cysteine spacing. No consensus site in the intracellular domain was found. Northern blotting of mouse and human tissues is in agreement with and expands those performed in rat and correlated well with the postulated presence of PV-1 in the endothelial diaphragms. The genomic organization of the human and mouse genes (HGMW-approved symbol PLVAP) has been determined, and the analysis of their 5' flanking regions has found a highly conserved classical TATA-driven promoter that shows several transcription factor consensus binding sites. Radiation hybrid panel mapping has localized the human and mouse PLVAP genes to chromosomes 19p13.2 and 8B3-C1, respectively.