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M J Bussemakers

Publications and source records attributed to M J Bussemakers.

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Genetic alterations in prostate cancer.

A number of genetic changes have been documented in prostate cancer, ranging from allelic loss to point mutations and changes in DNA methylation patterns (summarized in Fig. 1). To date, the most consistent changes are those of allelic loss events, with the majority of tumors examined showing loss of alleles from at least one chromosomal arm. The short arm of chromosome 8, followed by the long arm of chromosome 16, appear to be the most frequent regions of loss, suggesting the presence of novel tumor suppressor genes. Deletions of one copy of the Rb and p53 genes are less frequent, as are mutations of the p53 gene, and accumulating evidence suggests the presence of an additional tumor suppressor gene on chromosome 17p, which is frequently inactivated in prostate cancer. Alterations in the E-cadherin/alpha-catenin-mediated cell-cell adhesion mechanism appear to be present in almost half of all prostate cancers and may be critical to the acquisition of metastatic potential of aggressive prostate cancers. Finally, altered DNA methylation patterns have been found in the majority of prostate cancers examined, suggesting widespread alterations in methylation-modulated gene expression. The presence of multiple changes in these tumors is consistent with the multistep nature of the transformation process. Finally, efforts to identify prostate cancer susceptibility loci are under way and may elucidate critical early events in prostatic carcinogenesis.

Cadherins↗

Molecular biology of prostate cancer.

A number of genetic changes have been documented in prostate cancer, ranging from allelic loss to point mutations and changes in DNA methylation patterns (summarized in Fig 1). To date, the most consistent changes are those of allelic loss events, with the majority of tumors examined showing loss of alleles from at least one chromosomal arm. The short arm of chromosome 8, followed by the long arm of chromosome 16 appear to be the most frequent regions of loss, suggesting the presence of novel tumor suppressor genes. Deletions of one copy of the Rb and p53 genes are less frequent as are mutations of the p53 gene, and accumulating evidence suggests the presence of an additional tumor suppressor gene on chromosome 17p, which is frequently inactivated in prostate cancer. Alterations in the E-cadherin/alpha catenin mediated cell-cell adhesion mechanism appear to be present in almost half of all prostate cancers, and may be critical to the acquisition of metastatic potential of aggressive prostate cancers. Finally, altered DNA methylation patterns have been found in the majority of prostate cancers examined, suggesting widespread alterations in methylation-modulated gene expression. The presence of multiple changes in these tumors is consistent with the multistep nature of the transformation process. Finally, efforts to identify prostate cancer susceptibility loci are underway and will hopefully elucidate critical early events in prostatic carcinogenesis.

Cadherins↗

Homozygous deletion and frequent allelic loss of chromosome 8p22 loci in human prostate cancer.

Allelic loss studies have been instrumental in identifying tumor suppressor gene loci in a variety of cancers. In this study we analyzed prostate cancer specimens from 52 patients for allelic loss using 8 polymorphic probes for the short arm of chromosome 8. Overall, 32 of 51 (63%) informative tumors showed loss of at least one locus on chromosome 8p. The most frequently deleted region is observed at chromosome 8p22-8p21.2. Loss of one allele is identified in 14 of 23 (61%) tumors at D8S163, in 15 of 32 (47%) tumors at lipoprotein lipase, and in 20 of 29 (69%) tumors at MSR, all on 8p22. Loss of one allele is identified in 16 of 27 (59%) tumors at D8S220 at 8p21.3-8p21.2. In addition to frequent loss of one allele at the MSR locus, one metastatic prostate cancer sample demonstrated homozygous deletion of MSR sequences. Loci telomeric and centromeric to this region are largely retained. A chromosome 8p deletion map is constructed and defines the smallest region of overlap to a 14-cM interval at 8p22 between D8S163 and lipoprotein lipase, flanking the MSR locus. Evidence of chromosome 8q multiplication at locus D8S39 was detected in 5 of 32 (16%) tumors, all of which demonstrated loss with at least one probe on chromosome 8p. This study extends the previous finding of frequent loss of chromosome 8p in prostate cancer by defining a common region of loss of heterozygosity at 8p22 and a homozygous deletion of the MSR locus contained within this region. This is the first homozygous deletion identified in the genome of a human prostate cancer and the highest rate of loss yet reported on chromosome 8p in cancer. These results strongly suggest the presence of a tumor suppressor gene in this region which is frequently inactivated in prostate cancer.

Alleles↗

Molecular cloning and characterization of the human E-cadherin cDNA.

E-cadherin is a Ca(2+)-dependent cell adhesion molecule involved in cell-cell interaction. In its normal physiological function it plays an important role in embryonic development and tissue morphogenesis. Recent studies have shown that in cancer development E-cadherin can act as a suppressor of invasion. Indeed, in several kinds of carcinomas allelic loss of the E-cadherin/Uvomorulin locus and decreased E-cadherin expression have been described. The importance of E-cadherin in human cancer development may be substantiated by molecular analysis of the E-cadherin transcript. Therefore, we isolated and characterized the human E-cadherin cDNA. Comparison of the nucleotide and deduced amino acid sequences revealed that the human E-cadherin is highly homologous to the mouse E-cadherin (uvomorulin) and to other members of the cadherin family.

Amino Acid Sequence↗

Decreased expression of E-cadherin in the progression of rat prostatic cancer.

Cadherins represent a family of Ca(2+)-dependent cell adhesion molecules involved in homotypic, homophilic cell-cell interactions. Recent studies have shown that the cadherins can play a role in invasive and metastatic behavior. Using the established Dunning R-3327 model system of serially transplantable rat prostate cancers, the expression of E- and P-cadherin in rat prostatic cancer was studied. Analysis within this system demonstrated that whereas E-cadherin was expressed in the normal rat prostate and the well- or moderately differentiated, noninvasive Dunning tumors, no expression, either at the mRNA or at the protein level, could be detected in the invasive sublines. Since not all invasive Dunning tumors studied have metastatic ability, these results suggest that a decreased expression of E-cadherin is correlated with invasive behavior rather than with metastatic ability. Recently, genetic instability occurred in an animal bearing the well differentiated, androgen-responsive, slow growing, nonmetastatic Dunning R-3327-H rat prostate cancer resulting in the progression to an anaplastic, androgen-independent, fast growing, highly metastatic state. This spontaneously arising tumor, termed the AT6 subline, in its original host was heterogeneously composed of both a well differentiated and an anaplastic population of cancer cells in which areas of squamous cell differentiation were occasionally observed. The original animal bearing this heterogeneous AT6 cancer developed multiple metastases, the lung metastases being heterogeneously composed of anaplastic and squamous cell populations. Cytogenetic analysis demonstrated that the lung metastases were derived from a specific subpopulation of cancer cells present in the original AT6 primary tumor. Immunohistochemical studies demonstrated that only the area of lung metastases displaying squamous morphology were positive for E-cadherin. In contrast, the anaplastic areas of the lung metastases and the metastases in other organs were E-cadherin negative. By the first passage of the AT6 tumor only the anaplastic cells were present and no detectable E-cadherin mRNA or protein was found in the primary tumor and metastatic deposits. These results suggest that a decreased expression of E-cadherin is associated with the progression of prostatic cancer.

Animals↗

Differential expression of vimentin in rat prostatic tumors.

Differential hybridization analysis was used to identify molecular differences between a relative benign and a highly aggressive rat prostatic tumor derived from the Dunning R-3327-H adenocarcinoma. From the several differentially expressed mRNAs identified, we here report the characterization of pBUS51 which encodes a transcript highly expressed in all-anaplastic Dunning tumors. Only a very low expression was detectable in normal rat prostate or in the differentiated tumors of the Dunning system. Nucleotide sequence analysis and computer-assisted database comparison revealed that pBUS51 was highly homologous to vimentin and therefore likely the rat homolog of this protein.

Amino Acid Sequence↗

Increased expression of retroviral sequences in progressionally advanced rat prostatic tumors.

Differential hybridization analysis revealed two cDNA clones, pBUS19 and pBUS30, to be overexpressed in progressionally advanced rat prostatic tumors. Northern blot analysis suggested the clones to be related although no homology in nucleotide sequence could be shown. Isolation and characterization of a pBUS19-related clone, pJG116, and computer-assisted database comparison showed that all three clones could be mapped within a rat-specific endogenous retrovirus. The importance of overexpression of retroviral sequences in advanced prostatic cancer remains unclear.

Animals↗

Comparative structural analysis of the transcriptionally active proopiomelanocortin genes A and B of Xenopus laevis.

In the intermediate lobe of the pituitary gland, the prohormone proopiomelanocortin (POMC) is processed to, among other peptides, melanocyte-stimulating hormone (alpha-MSH). In the toad Xenopus laevis alpha-MSH controls skin darkening during background adaptation, and the level of POMC gene transcription in the intermediate lobe depends on the color of the background. In the lobe, two structurally different POMC proteins are produced from two mRNAs that are transcribed to approximately the same level from two POMC genes (A and B). We previously reported the entire nucleotide sequence of Xenopus POMC gene B. To identify conserved-- and thus potential regulatory--DNA elements in the Xenopus POMC gene, we here report the determination and analysis of the complete nucleotide sequence of Xenopus POMC gene A and its 5'- and 3'-flanking regions. Comparison of the two Xenopus POMC genes revealed, in addition to the exons, three highly conserved regions. First, the promoter regions are greater than 90% identical. The second region concerns JH12 repetitive elements situated at approximately the same position in both genes. These elements are greater than 86% identical. The third region is a 500-bp sequence just upstream of exon three (63% identity). Besides these three large regions, several small regions with significant identity were found at similar positions in the two POMC genes. The fact that, except for the JH12 element, the repetitive elements are not conserved between the two POMC genes indicates that these repeats are not functionally important.

Animals↗

Structural analysis of the entire proopiomelanocortin gene of Xenopus laevis.

In the pars intermedia of the pituitary the prohormone proopiomelanocortin (POMC) is tissue-specifically processed to, among other peptides, alpha-melanotropin (alpha MSH). In the South African clawed toad Xenopus laevis this hormone mediates the process of background adaptation: release of alpha-MSH causes darkening of the animal, while inhibition of alpha-MSH release results in a pale toad. Elevated release of alpha-MSH coincides with a higher rate of POMC gene transcription. The present study aims to find possible transcriptional regulatory elements in the Xenopus POMC gene. For that purpose the complete nucleotide sequence of the POMC gene and its 5'- and 3'- flanking regions were determined and analyzed. The Xenopus POMC gene promoter contains several regions which may be regulatory DNA elements in view of their similarity with corresponding regions of mammalian POMC gene promoters. In the rat POMC gene promoter, many of these regions represent protein-binding sequences. Besides the promoter sequence and the protein-coding sequences, no other segments with significant identity between the Xenopus and human POMC genes were found. Intron A of the Xenopus POMC gene contains a simple sequence, (TATC)76, and a JH12 repetitive element, while the 3'-flanking region contains a repetitive-EcoRI-monomer-2 element. Comparison of the JH12 sequence of the POMC gene with JH12 sequences from other Xenopus genes revealed a 335-bp consensus sequence which is flanked by a 30-bp inverted repeat. This JH12 consensus sequence is significantly larger than the previously reported JH12 core region. Alignment of intron B of the Xenopus POMC gene with database sequences revealed a consensus sequence of a novel Xenopus repetitive element of 330 bp flanked by a nearly perfect inverted repeat, indicating that this element may be a transposon-like element.

Animals↗

Identification of high mobility group protein I(Y) as potential progression marker for prostate cancer by differential hybridization analysis.

One of the major problems in the diagnosis of localized prostatic tumors is to predict the aggressiveness of an individual tumor, which is presumably associated with chance to progression. In an attempt to find molecular markers that are specific for aggressive prostatic cancer cells, we compared steady-state mRNA levels of progressionally related prostatic tumors. The Dunning R-3327-H subline, a relatively benign rat prostatic tumor, was compared to the therefrom derived highly aggressive MatLyLu tumor by differential hybridization analysis. The differential screening revealed 26 complementary DNA clones that detected transcripts overexpressed in MatLyLu. Upon further screening on the entire panel of Dunning R-3327 sublines, it appeared that three clones (pBUS1, pBUS19, and pBUS30), detected transcripts specifically expressed in metastatic rat prostatic tumors. The expression pattern of pBUS19 and pBUS30 suggested a relation between these complementary DNAs. Nucleotide sequence analysis, however, could not yet substantiate this. Computer-assisted comparison of the DNA sequences revealed the presence of rat long terminal repeat-like repetitive elements in pBUS19. The differential expression of repetitive elements in progressionally related tumors is interesting, yet similar findings have not been reported in human malignancies. Nucleotide sequence analysis of pBUS1 indicated that this clone is identical or related to high mobility group protein I(Y), a non-histone nuclear protein. From recent studies it appeared that this protein might be implicated in replication and/or transcription processes and is induced in fast proliferating/undifferentiated cells. The overexpression of high mobility group protein I(Y) correlates rather with metastatic ability than with growth rate; hence it may serve as a valuable marker to identify progressionally advanced prostate cancer cells.

Animals↗

Oncogene expression in prostate cancer.

Molecular biology has proven to be instrumental in studies on the onset and progression of cancer. Reasoning that tumorigenesis can be considered as a multistep process in which a normal cell gradually escapes from its delicately regulated growth pattern, one might describe this process in terms of gene expression. The group of genes that are good candidates for investigation in this context are the proto-oncogenes. Recent progress in oncogene research, has led us to believe that the group of oncogenes is much greater in number than the fifty so far characterized, since all growth factor-, growth factor receptor-, signal transducer- and transcription regulating genes have a potency to become oncogenic. Recessive genes, such as tumor suppressor genes might be equally relevant to the onset and progression of cancer. In this paper we confine ourselves to the role of protooncogenes in urological cancers, in particular prostate cancer. The usefulness of these genes as markers of progression in prostate cancer will be discussed.

Biomarkers, Tumor↗

The novel pituitary polypeptide 7B2 is a highly-conserved protein coexpressed with proopiomelanocortin.

In the amphibian intermediate pituitary gland the biosynthetic activity for production of the precursor protein proopiomelanocortin (POMC) can be physiologically manipulated; POMC synthesis is high in animals adapted to a black background and low in white-adapted animals. In order to study genes associated with POMC gene expression we applied a differential hybridization technique involving screening of a pituitary cDNA library with probes derived from RNA of inactive and physiologically activated intermediate pituitary cells of the amphibian Xenopus laevis. A differentially hybridizing Xenopus pituitary cDNA clone encoded the novel polypeptide 7B2. This Mr-21,000 secretory granule-associated protein of unknown function is shown to be highly conserved between Xenopus and human (83% amino acid sequence similarity). Conserved segments within the 7B2 structure included the N-terminal portion, three pairs of basic amino acids which are potential recognition sites for proteolytic enzymes, and three regions sharing similarity with putative GTP-binding domains. Levels of 7B2 mRNA were about 3% of POMC mRNA levels in Xenopus pituitary glands. In the intermediate pituitary the amount of both POMC and 7B2 mRNA was much higher in black-adapted toads than in white-adapted animals. These physiologically-induced changes in POMC and 7B2 mRNA levels were not found in the anterior pituitary. We conclude that the POMC and 7B2 genes are coexpressed and that modulation of the activity of these genes is tissue-specific.

Amino Acid Sequence↗

Down modulation of fibronectin messenger RNA in metastasizing rat prostatic cancer cells revealed by differential hybridization analysis.

To identify genes whose expression is down modulated in the process of metastasis, gene expression was analyzed in cell lines derived from Dunning R-3327 rat prostatic tumor sublines. A complementary DNA (cDNA) library from the anaplastic nonmetastasizing subline AT-1 was used for a differential hybridization analysis, using probes derived from mRNAs of the AT-1 and the metastasizing MAT-LyLu subline. In this way 14 cDNA clones were isolated representing 6 differentially expressed genes. The expression levels in a panel of tumor sublines measured with these cDNA clones were tested for correlation with the anaplastic non-metastasizing phenotype. One cDNA clone, designated pSE-1, whose expression was high in all tested sublines with that phenotype, appeared to represent the gene for fibronectin. To further investigate the down modulation of this gene, we studied its expression in AT-2 (anaplastic, nonmetastasizing tumor) and lines derived therefrom that exhibited a high metastatic potential after transfection with the v-Ha-ras oncogene. In the genetically manipulated metastasizing tumor sublines, fibronectin mRNA levels were approximately 4- to 8-fold lowered compared to the nonmetastasizing parental AT-2 line.

Animals↗

Expression of proto-oncogenes in xenografts of human renal cell carcinomas.

In a recent paper, we described the expression pattern of proto-oncogenes in primary human renal cell carcinoma [12]. To test the possibility of using xenografts as a useful alternative for such studies, we analyzed xenografts of a number of human renal cell carcinomas in nu/nu mice. Xenografts included RC2, RC14, RC21, RC43 and NC65. Northern blot analysis indicated that c-Ras was expressed in all these xenografts. The identity of the ras transcripts in the individual xenografts was further specified as c-Ha-ras, c-Ki-ras or N-ras. Expression of c-myc and the p53 gene was also found in a number of these tumors. Only RC21 failed to express the c-myc or the p53 gene. In all xenografts, a 3.0 kb c-fes/fps mRNA was present. In RC2, RC14, RC21 and RC43, low levels of the 4.8 kb ab 1 transcript were detectable. Transcripts of myb and sis could not be detected in any of the xenografts. The results indicated that the expression pattern of a variety of proto-oncogenes in xenografts of human renal cell carcinomas was similar to that in the primary tumors.

Animals↗

Characterization of human c-fes/fps reveals a new transcription unit (fur) in the immediately upstream region of the proto-oncogene.

Comparison of nucleotide sequence data of the 5' region of a fes/fps viral oncogene with those of the v-fes/fps homologous regions of man and cat revealed the position of the 3' portion of an as yet unidentified c-fes/fps exon. Comparative Southern blot and heteroduplex analysis of human and feline DNA immediately upstream of the v-fes/fps homologous regions showed extensive but discontinuous homology over a 9 kbp DNA stretch, which we have designated as fur. Northern blot analysis of mRNA from KG-1 myeloid cells with fes/fps- or fur-specific probes revealed a 3.0 kb fes/fps and a 4.5 kb fur transcript. Analysis of a number of tissues of an adult Wistar Lewis rat for the presence of fur transcripts revealed its differential expression pattern. An 0.95 kbp fes/fps-related and a 2.2 kbp fur-related cDNA recombinant clone were isolated from an oligo(dT)-primed KG-1 cDNA library. Comparative nucleotide sequence analysis of the fes/fps cDNA and its human genomic counterpart indicated that the cDNA contained genetic sequences that were identical to and colinear with exon 15-19 and, furthermore, that the poly(A) addition signal near the 3' end of exon 19 was functional. Similar analysis of the 2.2 kbp fur cDNA indicated that the poly(A) addition signal of the fur transcript was in close proximity of the newly discovered fes/fps exon. The region in between contained a CATT sequence but no 'TATA' box. The fur transcript was characterized by a long noncoding region at its 3' end.

Animals↗