Detection of chromosomal aberrations by comparative genomic hybridization during transformation of human breast epithelial cells in vitro.

Breast cancer is the most frequent malignancy in women. It is well recognized that tumorigenesis is a multistep process resulting from the accumulation of sequential genetic alterations. In breast cancers LOH has been described on one or both arms of multiple chromosomes. Comparative genomic hybridization (CGH) analysis was performed to identify chromosomal imbalances in the breast epithelial cells (HBEC). We have used a human in vitro-in vivo system in which the environmental carcinogen benz(a)pyrene (BP) and the c-Ha-ras oncogene were utilized for inducing in vitro transformation of HBEC. Immortal MCF-10F cells were treated with BP which resulted in the transformed cell line BP-1 that was further enhanced by transfection with the c-Ha-ras to generate the cell line BP-1-Tras. This cell line is tumorigenic when injected in severe combined immunodeficient (SCID) mice, generating the tumor cell line BP-1-Tras T J#4. Our comparative genomic hybridization analysis indicates that the most overrepresented segment after cell transformation and in the BP-1, BP-1-Tras and in the tumor cell line were 1p (80%), 5q21-ter (80%), 8q24.1 (90%) and Xq27-28 (60%). DNA sequence amplification at 10p14-15 was observed in BP-1-Tras T J#4 cells. Allelic losses of chromosome 4, 8p11-21 and 15q11-12, occur after cell transformation and are maintained consistently during tumorigenesis.

Neurofibromatosis 2 and malignant mesothelioma.

Mutations of the neurofibromatosis 2 (NF2) tumor suppressor gene cause the inherited disorder NF2 and are also common in malignant mesothelioma, which is not a characteristic feature of NF2. The authors report an asbestos-exposed person with NF2 and malignant mesothelioma. Immunohistochemical analysis of the mesothelioma confirmed loss of expression of the NF2 protein, and comparative genomic hybridization revealed losses of chromosomes 14, 15, and 22, and gain of 7. The authors propose that a person with a constitutional mutation of an NF2 allele is more susceptible to mesothelioma.

Loss of heterozygosity analysis defines a 3-cM region of 15q commonly deleted in human malignant mesothelioma.

Previous comparative genomic hybridization and allelic loss analyses demonstrated frequent deletions from 15q11.1-15 in malignant mesothelioma. Recurrent losses of 15q11-22 have also been reported in several other tumor types such as breast and colorectal cancers. To more precisely map the commonly deleted region, we have performed a high density loss of heterozygosity analysis of 46 malignant mesotheliomas, using 26 polymorphic microsatellite markers spanning the entire long arm of chromosome 15. Allelic loss from 15q was observed in 22 of 46 (48%) cases. These analyses have defined a minimally deleted region of approximately 3-cM, which was confirmed to reside at 15q15 by fluorescence in situ hybridization analysis with yeast artificial chromosome probes. No tumor suppressor genes have been reported to map to this site. The minimally deleted region identified in this investigation overlaps those observed in other kinds of cancer, and is the smallest site of recurrent 15q loss identified to date in human tumors. The identification of this commonly deleted site implicates a putative tumor suppressor gene(s) at 15q15 involved in diverse forms of human neoplasia.

Cloning and chromosomal localization of a gene encoding a novel serine/threonine kinase belonging to the subfamily of testis-specific kinases.

Using reverse transcription-polymerase chain reaction (RT-PCR) with degenerate oligonucleotides corresponding to two highly conserved motifs within the protein kinase family of catalytic domains, we isolated a PCR fragment encoding a novel member of the testis-specific serine/threonine kinases (STK) from mouse male mixed germ cell mRNA. This PCR fragment recognized a 1020-bp transcript in male germ cells by northern blot analysis and was used to clone a full-length cDNA from a mouse mixed germ cell cDNA library. This cDNA has an open reading frame of 804 bases encoding a protein of 268 amino acids. This novel gene is almost identical to Stk22c, encoding a recently described testis-specific protein kinase, except for base-pair deletions that result in a shift in the coding region and an alteration of 22 amino acids (residues 109-131). Due to its homology with Stk22c, we have called this protein kinase gene Stk22d. Northern blot analysis revealed that this protein kinase is developmentally expressed in testicular germ cells and is not present in brain, ovary, kidney, liver, or early embryonic cells. We then cloned the human homologue of this protein kinase gene (STK22C) and found it to be expressed exclusively in the testis. Fluorescence in situ hybridization with both the human and mouse cDNA clones revealed syntenic localization on chromosomes 1p34-p35 and 4E1, respectively.

Genomic imbalances in human lung adenocarcinomas and squamous cell carcinomas.

Comparative genomic hybridization analysis was performed on 67 non-small-cell lung cancers (NSCLCs), including 32 squamous cell carcinomas (SCCs) and 35 adenocarcinomas (ACs), to identify differences in the patterns of genomic imbalance between these two histologic subtypes. Among the entire tumor set, the chromosome arms most often overrepresented were 1q, 3q, 5p, and 8q, each detected in 50-55% of cases. The most frequently underrepresented arms were 9q, 3p, 8p, and 17p. The number of imbalances was similar in SCCs and ACs (median number/case: 12 and 11, respectively). Moreover, many imbalances, such as gains of 1q, 5p, and 8q, occurred at a high frequency in both histologic subgroups. Several statistically significant differences, however, were found. The most prominent difference was gain of 3q24-qter, seen in 81% of SCCs compared with 31% of ACs (P < 0.0001), with amplification at 3q25-26 being detected in eight of 32 (25%) SCCs but in only two of 35 (6%) ACs. Gain of 20p13 and loss of 4q also were seen at a significantly higher rate in SCCs than in ACs, whereas overrepresentation of 6p was more common in ACs. Gains of 7q and 8q each were associated with higher-stage tumors and either positive nodal involvement or higher tumor grade. These data suggest that genes located in several chromosomal regions, particularly 3q25-26, may be associated with phenotypic properties that differentiate lung SCCs from ACs. Furthermore, certain imbalances, prominent among them gains of 7q and 8q, may be indicative of tumor aggressiveness in NSCLCs.

Human hepatocellular carcinoma is characterized by a highly consistent pattern of genomic imbalances, including frequent loss of 16q23.1-24.1.

Comparative genomic hybridization (CGH) analysis was used to identify chromosomal imbalances in 52 human primary hepatocellular carcinomas (HCCs). The most prominent changes were gains of part or all of chromosome arms 8q (83% of cases) and 1q (73%) and loss of 16q (63%). Other commonly overrepresented sites were 5p, 7q, and Xq. Recurrent sites of DNA sequence amplification included 8q23--24 (five cases) and 11q13--14 (four cases). Other frequently underrepresented sites were 4q, 8p, 16p, and 17p. Taken collectively, these findings and data from other CGH studies of HCCs define a subset of chromosome segments that are consistently over- or underrepresented and highlight sites of putative oncogenes and tumor suppressor genes, respectively, involved in hepatocellular oncogenesis. Loss of heterozygosity analysis with a panel of polymorphic microsatellite markers distributed along 16q defined a minimal region of chromosomal loss at 16q23.1--24.1, suggesting that this region harbors a tumor suppressor gene whose loss/inactivation may contribute to the pathogenesis of many HCCs.

Identification and structural analysis of human RBM8A and RBM8B: two highly conserved RNA-binding motif proteins that interact with OVCA1, a candidate tumor suppressor.

The OVCA1 gene is a candidate for the breast and ovarian tumor suppressor gene at chromosome 17p13.3. To help determine the function(s) of OVCA1, we used a yeast two-hybrid screening approach to identify OVCA1-associating proteins. One such protein, which we initially referred to as BOV-1 (binder of OVCA1-1) is 173 or 174 amino acids in length and appears to be a new member of a highly conserved RNA-binding motif (RBM) protein family that is highly conserved evolutionarily. Northern blot analysis revealed that BOV-1 is ubiquitously expressed and that three distinct messenger RNA species are expressed, 1-, 3.2-, and 5.8-kb transcripts. The 1-kb transcript is the most abundant and is expressed at high levels in the testis, heart, placenta, spleen, thymus, and lymphocytes. Using fluorescence in situ hybridization and the 5.8-kb complementary DNA probe, we determined that BOV-1 maps to both chromosome 5q13-q14 and chromosome 14q22-q23. Further sequence analysis determined that the gene coding the 1- and the 3.2-kb transcripts (HGMW-approved gene symbol RBM8A) maps to 14q22-q23, whereas a second highly related gene coding for the 5.8-kb transcript resides at chromosome 5q13-q14 (HGMW-approved gene symbol RBM8B). The predicted proteins encoded by RBM8A and RBM8B are identical except that RBM8B is 16 amino acids shorter at its N-terminus. Molecular modeling of the RNA-binding domain of RBM8A and RBM8B, based on homology to the sex-lethal protein of Drosophila, identifies conserved residues in the RBM8 protein family that are likely to contact RNA in a protein-RNA complex. The conservation of sequence and structure through such an evolutionarily divergent group of organisms suggests an important function for the RBM8 family of proteins.

Functional roles of chromosomes 11 and 17 in the transformation of human breast epithelial cells in vitro.

Genomic alterations in primary breast cancer play a role in the initiation and progression of the disease. We have analyzed the molecular events involved in the initiation and progression of the neoplastic process in an in vitro experimental system. Immortalization of human breast epithelial cells (HBEC) is associated with 3:9 translocation, p53 mutation and microsatellite instability (MSI) of chromosomes 11p13, and 17p. BP1-E cells, derived from the immortalized MCF-10F cells transformed by the carcinogen benzo(a)pyrene (BP), express in vitro growth advantage, anchorage independence, enhanced chemoinvasiveness, loss of ductulogenic capabilities and tumorigenesis in a heterologous host. This neoplastic progression is also associated with mutations and/or amplification of c-H-ras, int-2, c-neu, c-myc and MDM2, MSI at 11q25 and 13q12-q13 and loss of heterozygosity at 17p. In order to test whether chromosomes 11 or 17 play a functional role in the phenotypic expression of transformation of BP1E cells, we utilized microcell-mediated chromosome transfer (MMCT) technique for inserting the corresponding normal chromosomes to these transformed cells. BP1E cells were transfected with PsV2neo plasmid and fused with microcells obtained from the mouse cell line A9, containing a normal chromosome 11 or 17 (A9-11neo and A9-17neo cells, selected in G418 and cloned. Sixteen primary microcell hybrids from each chromosome transfer, designated BP1E-11neo and BP1E-17neo survived selection in G-418 containing medium. A single clone from each group, BP1E-11neo #145 and BP1E-17neo D100, survived subcloning and were utilized for a detailed panel of analyses. The presence of a donor chromosome was confirmed by dual color fluorescence in situ hybridization (FISH), southern blot analysis of the marker vector pSV2neo, and microsatellite polymorphism analysis. The transfer of the normal chromosomes 11 and 17 resulted in a 50% and 90% inhibition of cell growth respectively, and reduced both colony efficiency and colony size. Telomerase activity was significantly reduced only by chromosome 17 insertion, providing a possible explanation for the more significant senescence observed in BP1E-17neo D100 cells. Microsatellite polymorphism analysis revealed that three loci, 11q13-23, 11q23.1, and 11q23.3 (markers D11S911, DRD2, and D11S29) were retained in BP1E-11neo #145 cells, and two, 17q24.2-25.2, 17q25.2 (markers D17S515 and D17S785 were retained in BP1E-17neo D100 cells. We conclude that the specific regions of normal chromosomes 11 and 17 transferred play a functional role in the expression of immortal and transformed phenotypes of HBEC in vitro.

Novel human and mouse annexin A10 are linked to the genome duplications during early chordate evolution.

We have identified and characterized a 12th subfamily of vertebrate annexins by systematic analysis of the primary structure, chromosomal mapping, and molecular evolution of unique cDNA and protein sequences from human and mouse. Distinctive features included rare expression, a codon deletion in conserved repeat 3, and an unusual ablation of the type II calcium-binding sites in tetrad core repeats 1, 3, and 4. The paralogy of novel annexin A10 (following revised nomenclature) was confirmed by FISH-mapping human ANXA10 to chromosome 4q33 and genetic linkage mapping mouse Anxa10 to midchromosome 8. Phylogenetic analysis established that the 5' and 3' halves of the annexin A6 octad are more closely related to annexins A5 and A10, respectively, than they are to each other. Molecular date estimates, paralogy linkage maps between human chromosomes 4 and 5, and annexin structural considerations led to the proposal that annexins A5 and A10 may have been the direct progenitors of annexin A6 octad formation via chromosomal duplication during the genome expansion in early chordates.

Comparative genomic hybridization and loss of heterozygosity analyses identify a common region of deletion at 15q11.1-15 in human malignant mesothelioma.

Comparative genomic hybridization analysis was performed to identify chromosomal imbalances in 24 human malignant mesothelioma (MM) cell lines derived from untreated primary tumors. Chromosomal losses accounted for the majority of genomic imbalances. The most frequent underrepresented segments were 22q (58%) and 15q1.1-21 (54%); other recurrent sites of chromosomal loss included 1p12-22 (42%), 13q12-14 (42%), 14q24-qter (42%), 6q25-qter (38%), and 9p21 (38%). The most commonly overrepresented segment was 5p (54%). DNA sequence amplification at 3p12-13 was observed in two cases. Whereas some of the regions of copy number decreases (i.e., segments in 1p, 6q, 9p, and 22q) have previously been shown to be common sites of karyotypic and allelic loss in MM, our comparative genomic hybridization analyses identified a new recurrent site of chromosomal loss within 15q in this malignancy. To more precisely map the region of 15q deletion, loss of heterozygosity analyses were performed with a panel of polymorphic microsatellite markers distributed along 15q, which defined a minimal region of chromosomal loss at 15q11.1-15. The identification of frequent losses of a discrete segment in 15q suggests that this region harbors a putative tumor suppressor gene whose loss/inactivation may contribute to the pathogenesis of many MMs.

Characterization of MOAT-C and MOAT-D, new members of the MRP/cMOAT subfamily of transporter proteins.

BACKGROUND: Multidrug resistance-associated protein (MRP) and canalicular multispecific organic anion transporter (cMOAT) are transporter proteins that pump organic anions across cellular membranes and have been linked to resistance to cytotoxic drugs. We previously identified MOAT-B, an MRP/cMOAT-related transporter, by use of a polymerase chain reaction approach. However, analysis of expressed sequence tag (EST) databases indicated that there might be additional MRP/cMOAT-related transporters. To further define the MRP/cMOAT subfamily of transporters, we used EST probes to isolate complementary DNAs for two related transporter proteins, MOAT-C and MOAT-D. METHODS: MOAT-C and MOAT-D expression patterns in human tissues were determined by RNA blot analysis, and chromosomal localization of the genes was determined by fluorescence in situ hybridization. RESULTS: MOAT-C is predicted to encode a 1437-amino-acid protein that, among eukaryotic transporters, is most closely related to MRP, cMOAT, and MOAT-B (about 36% identity). However, MOAT-C is less related to MRP and cMOAT than MRP and cMOAT are to each other (about 48% identity). Like MOAT-B, MOAT-C lacks an N-terminal membrane-spanning domain, indicating that the topology of this protein is similarly distinct from that of MRP and cMOAT. MOAT-D is predicted to encode a 1527-amino-acid protein that is the closest known relative of MRP (about 58% identity). MOAT-D is also highly related to cMOAT (about 47% identity). The presence of an N-terminal membrane-spanning domain indicates that the topology of MOAT-D is quite similar to that of MRP and cMOAT. MOAT-C transcripts are widely expressed in human tissues; however, MOAT-D transcript expression is more restricted. The MOAT-C and MOAT-D genes are located at chromosomes 3q27 and 17q21.3, respectively. CONCLUSIONS: On the basis of amino acid identity and protein topology, the MRP/cMOAT transporter subfamily falls into two groups; the first group consists of MRP, cMOAT, and MOAT-D, and the second group consists of MOAT-B and MOAT-C.

Combined chromosome microdissection and comparative genomic hybridization detect multiple sites of amplification DNA in a human lung carcinoma cell line.

Chromosome microdissection-fluorescence in situ hybridization and comparative genomic hybridization (CGH) were performed in parallel to identify the native location of amplified DNA in a human non-small cell lung cancer (NSCLC) cell line exhibiting a homogeneously staining region (hsr) and double minutes (dmin). The native locations of microdissected DNA from the hsr and dmin were 7p12-13 and 8q24, respectively. Southern analysis revealed coamplification of EGFR (7p12) and MYC (8q24). CGH detected amplification of DNA not only from 7p12-13 and 8q24, but also from 9p24 and 10q22.

Characterization of unconventional MYO6, the human homologue of the gene responsible for deafness in Snell's waltzer mice.

Deafness is the most common form of sensory impairment in humans. Mutations in unconventional myosins have been found to cause deafness in humans and mice. The mouse recessive deafness mutation, Snell's waltzer, contains an intragenic deletion in an unconventional myosin, myosin VI (locus designation, Myo6). The requirement for Myo6 for proper hearing in mice makes this gene an excellent candidate for a human deafness disorder. Here we report the cloning and characterization of the human unconventional myosin VI (locus designation, MYO6) cDNA. The MYO6 gene maps to human chromosome 6q13. The isolation of the human gene makes it now possible to determine if mutations in MYO6 contribute to the pathogenesis of deafness in the human population.

Comparative genomic hybridization analysis detects frequent, often high-level, overrepresentation of DNA sequences at 3q, 5p, 7p, and 8q in human non-small cell lung carcinomas.

Comparative genomic hybridization analysis was used to identify chromosomal imbalances in 20 non-small cell lung carcinoma (NSCLC) biopsies and cell lines. The chromosome arms most often overrepresented were 3q (85%), 5p (70%), 7p (65%), and 8q (65%), which were observed at high copy numbers in many cases. Other common overrepresented sites were 1q, 2p, and 20p. DNA sequence amplification was often observed, with the most frequent site being 3q26 (six cases). Other recurrent sites of amplification included 8q24, 3q13, 3q28-qter, 7q11.2, 8p11-12, 12p12, and 19q13.1-13.2. The most frequent underrepresented segment was 3p21 (50%); other recurrent sites of autosomal loss included 8p21-pter, 15q11.2-13, 5q11.2-15, 9p, 13q12-14, 17p, and 18q21-qter. These regions of copy number decreases are also common sites of allelic loss, further implicating these sites as locations of tumor suppressor genes. Although some of the overrepresented segments harbor known or suspected oncogenes/growth-regulatory genes, we have identified 3q and 5p as new sites that are very frequently overrepresented in NSCLC. These findings could represent entry points for the identification of novel amplified DNA sequences that may contribute to the development or progression of NSCLC.

Advances in the analysis of chromosome alterations in human lung carcinomas.

A review of chromosomal analyses of human lung carcinomas is presented. Karyotypic studies have revealed multiple cytogenetic changes in most small cell lung carcinomas (SCLCs) and non-small cell lung carcinomas (NSCLCs). In SCLCs, losses from 3p, 5q, 13q, and 17p predominate; double minutes associated with amplification of members of the MYC oncogene family may be common late in disease. In NSCLCs, deletions of 3p, 9p, and 17p, +7, i(5)(p10), and i(8)(q10) often are reported. The recurrent deletions encompass sites of tumor suppressor genes commonly inactivated in lung carcinomas, such as CDKN2 (9p21), RB1 (13q14), and TP53 (17p13). Despite technical advances in cell culture, the rate of successful karyotypic analysis of lung carcinomas has remained low. Alternative molecular cytogenetic methods to assess chromosome changes in lung cancer, particularly comparative genomic hybridization (CGH) analysis, are discussed. Initial CGH studies confirm the existence of many of the karyotypic imbalances identified earlier in lung cancer and have revealed several recurrent abnormalities, such as 10q- in SCLC, that had not been recognized previously. The further application of such molecular cytogenetic approaches should enable investigators to define more precisely the spectrum and clinical implications of chromosome alterations in lung cancer.

Loss of heterozygosity analysis defines a critical region in chromosome 1p22 commonly deleted in human malignant mesothelioma.

Previous cytogenetic analysis has revealed frequent losses of chromosome 1p21-22 in human malignant mesothelioma, suggesting that the loss or inactivation of a tumor suppressor gene's) residing at this site may contribute to the tumorigenic conversion of mesothelial cells. To more precisely define the location of the target gene, primary tumor specimens and cell lines from 50 malignant mesotheliomas were examined for loss of heterozygosity using short tandem repeat polymorphism (STRP) markers. Nineteen STRP markers established by the Cooperative Human Linkage Center were selected for the initial screening of the entire short arm of chromosome 1. Thirty-seven cases (74%) showed allelic losses at least at one locus in 1p. Thirty-six of these cases showed losses of 1p21-22, including 23 with partial deletions involving this region. To obtain a higher resolution map of this region, another 13 STRP markers from the Genethon map were used to define the shortest region of overlapping deletions to a 4-cM segment flanked by the loci D1S435 and D1S236. The chromosomal location of the critically deleted region was confirmed to be within 1p22 by karyotypic and fluorescence in situ hybridization analyses.

Cytogenetic studies on a patient with prepubertal breast cancer: a case report.

Cytogenetic studies were carried in a 10 year old girl with prepubertal breast cancer for assessing inherited genetic susceptibility to chromosome breakage. The girl presented with a tumour in the left breast. Histologically it was diagnosed as secretory carcinoma (SC). Chromosome anomalies observed in phytohemagglutinin (PHA-P) stimulated lymphocytes were del(2)(q33), del(3)(p24), del(7)(q22) and dup(12)(p11p12). The regions involved have been reported in breast tumors. These loci, detected in peripheral blood lymphocytes (PBL), could be the sites susceptible to breakage, its subsequent effect being manifested in the target (breast) tissue.

Cytogenetic studies on patients of acute lymphoblastic leukemia Burkitt's type with (8;14) & (14;18) translocations.

Eight patients with acute lymphoblastic leukemia of Burkitt's type (ALL-L3) and two patients with Burkitt's lymphoma (BL) were subjected for cytogenetic studies. Translocation (8;14)(q24;q32) was present in nine (90%) patients; seven patients of ALL-L3 and two of BL. One ALL-L3 patient revealed t(14;18)(q32;q21) in 100 per cent metaphases. Additional clonal chromosomal anomalies present in these patients were deletion (6q) (40%) and trisomy 21(20%). The occurrence of t(8;14)(q24;q32) in ALL-L3 and BL patients in our series supports the association of t(8;14) with ALL-L3 and Burkitt's lymphoma.

Flow cytometric DNA analysis of squamous cell carcinomas of the oral cavity: correlation with clinical and histopathological features.

DNA content was measured in 68 squamous cell carcinomas (SCC) of the oral cavity by flow cytometry. Samples fixed in 95% alcohol and disaggregated with 0.5% pepsin were stained with 4,6-diamidino-phenylindole (DAPI) for flow cytometry. The tumours were classified according to the TNM classification--1987, and graded histopathologically. A positive correlation between tumour size and ploidy status was observed. Poorly differentiated tumours were mainly non-diploid (P < 0.01, chi 2). A majority of the node positive (N+) tumours were non-diploid (P < 0.05). It was possible to distinguish diploid, N+, T4 tumours from diploid, N-, T4 tumours by their higher S-phase fraction (SpF). SpF was also a useful parameter to differentiate diploid, N+ tumours from diploid, N- tumours among the moderately differentiated (MD) SCC. These results suggest that ploidy and SpF can be useful correlates of tumour behaviour.