Localization of HeLa cell tumor-suppressor gene to the long arm of chromosome II.

Cytogenetic and molecular genetic analyses of human intraspecific HeLa x fibroblast hybrids have provided evidence for the presence of a tumor-suppressor gene(s) on chromosome 11 of normal cells. In the present study, we have carried out extensive RFLP analysis of various nontumorigenic and tumorigenic hybrids with at least 50 different chromosome 11-specific probes to determine the precise location of this tumor-suppressor gene(s). Two different hybrid systems, (1) microcell hybrids derived by the transfer of a normal chromosome 11 into a tumorigenic HeLa-derived hybrid cell and (2) somatic cell hybrids derived by the fusion of the HeLa (D98OR) cells to a retinoblastoma (Y79) cell line, were particularly informative. The analysis showed that all but one of the nontumorigenic hybrid cell lines contained a complete copy of the normal chromosome 11. This variant hybrid contained a segment of the long arm but had lost the entire short arm of the chromosome. The tumorigenic microcell and somatic cell hybrids had retained the short arm of the chromosome but had lost at least the q13-23 region of the chromosome. Thus, these results showed a perfect correlation between the presence of the long arm of chromosome 11 and the suppression of the tumorigenic phenotype. We conclude therefore that the gene(s) involved in the suppression of the HeLa cell tumors is localized to the long arm (q arm) of chromosome 11.     

Deletion and translocation of chromosome 11q13 sequences in cervical carcinoma cell lines.

Molecular genetic studies on HeLa cell-derived nontumorigenic and tumorigenic hybrids have previously localized the HeLa cell tumor-suppressor gene to the long arm of chromosome 11. Extensive molecular and cytogenetic studies on HeLa cells have shown chromosome band 11q13 to be rearranged in this cell line. To determine whether q13 rearrangement is a nonrandom event in cervical carcinomas, six different human papilloma virus (HPV)-positive (HeLa, SiHa, Caski, C4-I, Me180, and Ms751) and two different HPV-negative (C33A and HT3) cell lines were studied. Long-range restriction mapping using a number of q13-specific probes showed molecular rearrangements within 75 kb of INT2 probe in three HPV-positive cell lines (HeLa, SiHa, and Caski) and in an HPV-negative cell line (HT3). FISH using an INT2 YAC identified a breakpoint within the sequences spanned by this YAC in two of the cell lines, HeLa and Caski. INT2 cosmid derived from this YAC showed deletion of cosmid sequences in two other cell lines, SiHa and C33A. These two cell lines, however, retained cosmid sequences of Cyclin D1, a probe localized 100 kb proximal to INT2. Deletions being the hallmark of a tumor-suppressor gene, we conclude that the 100-kb interval between the two cosmids might contain sequences of the cervical carcinoma tumor-suppressor gene.     

Transfer of a normal human chromosome 11 suppresses tumorigenicity of some but not all tumor cell lines

The complete suppression of tumorigenicity of a human cervical cancer cell (HeLa) and a Wilms' tumor cell line (G401) following the introduction via microcell fusion of a single chromosome t(X;11) has been demonstrated by Stanbridge and co-workers. To determine whether other tumor cell lines are suppressed by chromosome 11, we performed chromosome transfer experiments via microcell fusion into various human tumor cell lines, including a uterine cervical carcinoma (SiHa), a rhabdomyosarcoma (A204), a uterine endometrial carcinoma (HHUA), a renal cell carcinoma (YCR-1), and a rat ENU-induced nephroblastoma (ENU-T1). We first isolated a mouse A9 cell containing a single human chromosome 11 with integrated pSV2-neo plasmid DNA. Following microcell fusion of the neo-marked chromosome 11 with the various tumors mentioned above, we isolated clones that were resistant to G418 and performed karyotypic analyses and chromosomal in situ hybridization to ensure the transfer of the marked chromosome. Whereas the parental cells of each cell line were highly tumorigenic, SiHa and A204 microcell hybrid clones at early passages were nontumorigenic in nude mice and HHUA was moderately tumorigenic. On the other hand, YCR-1 and ENU-T1 microcell hybrid clones were still highly tumorigenic following the introduction of chromosome 11. Thus, the introduction of a normal chromosome 11 suppresses the tumorigenicity of some but not all tumors, suggesting that the function of the putative suppressor gene(s) on chromosome 11 is effective only in specific tumors.     

Complementation of a DNA repair deficiency in six human tumor cell lines by chromosome 11

Summary Human tumor cells, after x-irradiation during the G₂ phase of the cell cycle, show an abnormally high frequency of persistent chromatid breaks and gaps resulting from deficient DNA repair. Addition of a single human chromosome 11 from normal fibroblasts by micro-cell fusion to cell lines from six different tumors resulted in efficient repair of the radiation-induced damage to the level in normal cells. For one of the cell lines, addition of the long arm of chromosome 11 was sufficient to restore repair efficiency. In four of the six tumor lines, restoration of efficient DNA repair by chromosome 11 was associated with tumor suppression in nude mice. These results suggest that chromosome 11 carries a DNA repair gene or genes that complement the repair deficiency of tumor cells and that this gene for at least one tumor is localized to the long arm.     

Localization of Human Cadherin Genes to Chromosome Regions Exhibiting Cancer-Related Loss of Heterozygosity

This report presents the chromosomal localization of cadherin genes. Cadherins are cellular adhesion molecules. Since disturbance of intracellular adhesion is important for invasion and metastasis of tumor cells, cadherins are considered prime candidates for tumor suppressor genes. A variety of solid tumors show loss of heterozygosity of the long arm of chromosome 16, which is indicative of the potential location of tumor suppressor genes. Refined and new localizations of six cadherin genes (CDH3, 5, 8, 11, 13, and 15) to the long arm of chromosome 16 are shown. CDH15 was localized to 16q24.3, in a region that exhibits loss of heterozygosity in a number of sporadic breast cancer tumors. Previous localization of CDH13 (H-cadherin) to 16q24 suggested this gene as a tumor suppressor candidate in the 16q24.3 loss of heterozygosity region; however, refined mapping presented in this report localizes CDH13 proximal to this region. A human EST homologous to the chicken cadherin-7 was partially sequenced and found to represent a new human cadherin. This cadherin mapped to chromosome 18q22–q23, a region that exhibits loss of heterozygosity in head and neck squamous cell carcinomas. CDH16 was localized to 8q22.1, a region exhibiting loss of heterozygosity in adult acute myeloid leukemia.     

Cervical carcinoma progression-associated genetic alterations on chromosome 6

To identify the loci associated with progression of cervical carcinoma, chromosome 6 regions were tested for loss of heterozygosity. Detailed analysis with 28 microsatellite markers revealed a high frequency of allelic deletions for several loci of the short (6p25, 6p22, 6p21.3) and long (6q14, 6q16-21, 6q23-24, 6q25, 6q27) arms of chromosome 6. Examination of 37 microdissected carcinoma and 22 cervical dysplasia specimens revealed allelic deletions from the HLA class I-III genes (6p22-21.3) and subtelomeric locus 6p25 were found in more than 40% dysplasia specimens. With multiple microdissection of cryosections, genetic heterogeneity of squamous cervical carcinoma was analyzed, and clonal and subclonal allelic deletions from chromosome 6 were identified. Half of the tumors had clonal allelic deletion of D6S273 (6p21.3), which is in a Ly6G6D (MEGT1) intron in the HLA class III gene locus. The frequency of allelic deletions from the chromosome 6 long arm was no more than 20% in dysplasias. Allelic deletions from two loci, 6q14 and 6q16-21, were for the first time associated with invasion and metastasis in cervical carcinoma.     

Karyotype pecularities of human colorectal adenocarcinomas

Summary The data of the chromosome abnormalities in 15 colorectal tumors are presented. Rearrangements of the short arm of chromosome 17, leading to deletions of this arm or its part were noted in 12 tumors; in 2 other cases, one of the homologs of pair 17 was lost. The losses of at least one homolog of other chromosomal pairs were also found: chromosome 18, in 12 out of 13 cases with fully identified numerical abnormalities; chromosome 5, in 6 tumors; chromosome 21, in 5 cases; chromosomes 4, 15, and 22, in 4 cases each. Additional homologs of pair 20 were observed in 6 tumors, extra 8q was found in 5 tumors, and extra 13q in 6 cases. Rearrangements of the short arm of chromosome 1 and the long arm of chromosome 11 characterized 6 tumors each. The data recorded in our series differ from the data of other authors in two respects: the high incidence of the loss of sex chromosomes and the rearrangements of the long arm of chromosome 9. X chromosomes were missing in 4 out of 7 tumors in females, and Y chromosomes were absent in 5 out of 8 tumors in males. The long arm of chromosome 9 was rearranged in 8 cases, in 5 of them the breakpoint being at 9q22. Cytological manifestations of gene amplification (double minutes or multiple microchromosomes) were noted in 6 tumors.     

Molecular analysis of the chromosome 11p region in renal cell carcinomas.

Comparative histological data suggest that papillary renal cell tumors in adults and Wilms' tumors in children develop from maturation-arrested cells of similar origin. Wilms' tumor is characterized by genetic changes at the chromosome 11p region. In the present study, we have analyzed 10 papillary and 10 non-papillary renal cell tumors and determined the allelic status of 6 loci on the short arm of chromosome 11. Only one papillary renal cell carcinoma among the 20 tumors showed a loss of constitutional heterozygosity for the chromosome 11p region. These data suggest that separate molecular events occur in the development of Wilms' tumor and papillary renal cell tumors, subsequent to the proliferation of maturation-arrested cells of the kidney.     

Genomic changes and HPV type in cervical carcinoma

To identify chromosomal regions that may include the loci of abnormally expressed cellular genes and may be specifically altered depending on the histological subtype of the tumor, we studied primary cervical carcinoma using CGH and HPV genotyping. Eighty-seven percent of the primary tumors were positive for DNA of a "high-risk" HPV type (e.g., 16 or 18). In the cervical carcinomas, without reference to histologic subtype, overrepresentation of chromosome 3q was the most consistent chromosomal aberration with underrepresentation of chromosome 3p also a frequent finding. Chromosome arms 1q, 5p, 20q, and Xq were overrepresented in many tumors and 3p loss and 5p, 8q, and 16q gain were only associated with squamous cell carcinoma in this series.     

Assignment of the pepsinogen gene complex (PGA) to human chromosome region 11q13 by in situ hybridization

The genes coding for human pepsinogen (PGA3, PGA4, and PGA5) were assigned to chromosome region 11q13 by in situ hybridization. Previously we localized the PGA gene complex to a centromeric region of chromosome 11 (p11----q13) by Southern blot analysis of mouse-human somatic cell hybrids. Our in situ hybridization results confirm this assignment and further localize the genes to a smaller region on the long arm.     

Identification of a human chromosome 11 gene which is differentially regulated in tumorigenic and nontumorigenic somatic cell hybrids of HeLa cells.

The tumorigenicity of HeLa cells in nude mice can be suppressed by the addition of a normal human chromosome 11 in somatic cell hybrids. We have attempted to identify specific genes involved in this phenomenon by transfecting a complementary DNA expression library into a tumorigenic HeLa-fibroblast hybrid. A cell line designated F2 was isolated which displayed morphological features of the nontumorigenic hybrids, demonstrated reduced tumorigenicity in nude mice, and showed an 85% reduction in alkaline phosphatase, a consistent marker of the tumorigenic phenotype in these cells. F2 contained a single exogenous complementary DNA, which was recovered by polymerase chain reaction and designated HTS1 because of its potential association with "HeLa tumor suppression." Northern blot studies suggested differential regulation of the HTS1 gene dependent on the tumorigenicity of the cell. In nontumorigenic hybrids, RNA species of 2.8, 3.1, and 4.6 kilobases were identified. In two tumorigenic hybrid lines, the 2.8-kilobase species was markedly reduced or absent. Similarly, three nontumorigenic human keratinocyte lines expressed all three RNA species, whereas several tumorigenic cervical carcinoma cell lines lacked the 2.8-kilobase species. Chromosome localization studies mapped the HTS1 gene to chromosome 11p15, a region of chromosome 11 that is believed to contain a tumor suppressor gene. These findings indicate that HTS1 represents a novel chromosome 11 gene which may be a target of the tumor suppressor gene active in this system.     

Assignment of the human stromelysin 3 (STMY3) gene to the q11.2 region of chromosome 22.

The human stromelysin 3 (STMY3) gene, a new member of the matrix metalloproteinase (MMP) gene family, may contribute to breast cancer cell invasion, and has been localized by in situ hybridization to the long arm of chromosome 22. As demonstrated using a panel of somatic cell hybrids, the STMY3 gene is in band 22q11.2, in close proximity to the BCR gene involved in chronic myeloid leukemia, but far from the (11;22) translocation breakpoint observed in Ewing sarcoma. This position differs from that reported on chromosomes 11 and 16 for the other MMP genes, suggesting that stromelysin 3 could be a member of a new MMP subfamily.     

Mapping of the versican proteoglycan gene (CSPG2) to the long arm of human chromosome 5 (5q12-5q14).

Versican is a major chondroitin sulfate proteoglycan of vascularized connective tissues whose eponym reflects its functional versatility in macromolecular affinity and interactions. In this report we have localized the versican gene (CSPG2) to the long arm of human chromosome 5 by utilizing a combination of somatic cell hybrids, Southern blotting, polymerase chain reaction, and chromosomal in situ hybridization. The proteoglycan gene segregated concordantly with hybrid cell lines containing the long arm of chromosome 5, comprising the 5q12-q14 band regions. To refine this locus further, we screened a chromosome 5-specific library and isolated several genomic clones encoding a portion of the 5' end of versican. One of these genomic clones was used as a probe for in situ hybridization of human chromosome metaphases. The results corroborated the data obtained using somatic cell hybrids and further refined the assignment of the versican gene to the narrow band region of 5q12-5q14, with the primary site likely to be 5q13.2. The availability of novel genomic clones and the mapping data presented here will make possible the identification of any defect genetically linked to this proteoglycan gene.     

Recurrent 1;17 translocations in human neuroblastoma reveal nonhomologous mitotic recombination during the S/G2 phase as a novel mechanism for loss of heterozygosity.

Neuroblastomas often show loss of heterozygosity of the chromosomal region 1p36 (LOH 1p), probably reflecting loss of a tumor-suppressor gene. Here we describe three neuroblastoma tumors and two cell lines in which LOH 1p results from an unbalanced translocation between the p arm of chromosome 1 and the q arm of chromosome 17. Southern blot and cytogenetic analyses show that in all cases the chromosome 17 homologue from which the 1;17 translocation was derived is still present and intact. This suggests a model in which a translocation between the short arm of chromosome 1 and the long arm of chromosome 17 takes place in the S/G2 phase of the cell cycle and results in LOH 1p. Nonhomologous mitotic recombination in the S/G2 phase is a novel mechanism of LOH.     

A gene for autosomal dominant nonsyndromic hearing loss (DFNA12) maps to chromosome 11q22-24.

We performed linkage analysis in a Belgian family with autosomal dominant midfrequency hearing loss, which has a prelingual onset and a nonprogressive course in most patients. We found LOD scores >6 with markers on chromosome 11q. Analysis of key recombinants maps this deafness gene (DFNA12) to a 36-cM interval on chromosome 11q22-24, between markers D11S4120 and D11S912. The critical regions for the recessive deafness locus DFNB2 and the dominant locus DFNA11, which were previously localized to the long arm of chromosome 11, do not overlap with the candidate interval of DFNA12.     

Molecular detection of a Yp/18 translocation in a 45,X holoprosencephalic male

Summary Prenatal diagnosis in a fetus with holoprosencephaly showed a 45,X karyotype and a suspected 18p abnormality. At birth, the fetus presented with normal male genitalia. Y chromatin was not cytogenetically detectable by Q-, G-, or G11-banding. Mosaicism for a cell line containing a Y chromosome was not observed in amniocytes, lymphocytes, or skin fibroblasts. Southern blot analysis for 11 different Y-DNA loci demonstrated the presence in the patient's genome of sequences derived from the short arm, centromeric region, and proximal long arm of the Y chromosome (intervals 1–5). The distal long arm of the Y (intervals 6 and 7) was absent. In situ hybridization with the Y-derived probe pDP105 showed silver grains over the short arm of the del(18) chromosome, suggesting a Y/18 translocation with loss of 18p and distal Yq material.     

Integration sites of human papillomavirus 18 DNA sequences on HeLa cell chromosomes

Human papillomaviruses (HPV) 16 and 18 are closely linked with human genital cancer. In most cervical carcinomas, viral sequences are integrated into the host genome. HeLa, a cervical carcinoma cell line, has multiple copies of integrated HPV 18 DNA. In this study, in situ chromosome hybridization was used to assign the integration sites of HPV 18 DNA sequences on HeLa cell chromosomes. Four sites of hybridization were identified at 8q23----q24, 9q31----q34, p11----p13 on an abnormal chromosome 5, and q12----q13 on an abnormal 22. Three of these sites correspond with the locations of MYC, ABL, and SIS protooncogenes, and are at or in close proximity to fragile sites. The chromosomal localization of HPV 18 DNA may be useful in assessing the role of viral integration in the development of this malignancy.     

Regional chromosome mapping of the human skin type I procollagen gene using adenovirus 12-fragmentation of human-mouse somatic cell hybrids

Prevous work, using human-mouse somatic cell hybrids, has localized the structural gene for human skin type I procollagen (COL 1) to chromosome 17. One of these hybrids contained only the long arm of human chromosome 17, translocated onto a mouse chromosome, as human chromosomal material. This hybrid was treated with adenovirus 12, and various clones were picked which contained different-sized fragments of human chromosome 17 that were still translocated onto a mouse chromosome. Measurements of these fragments, combined with assays for human COL 1 production and galactose kinase (GAK) activity (also localized on the long arm of human chromosome 17), has allowed us to regionally map the structural gene for human COL 1 to an area just distal to the thymidine kinase (TK) and GAK genes within bands q21 and q22 on human chromosome 17.     

An alternative route for multistep tumorigenesis in a novel case of hereditary renal cell cancer and a t(2;3)(q35;q21) chromosome translocation.

Through allele-segregation and loss-of-heterozygosity analyses, we demonstrated loss of the translocation-derivative chromosome 3 in five independent renal cell tumors of the clear-cell type, obtained from three members of a family in which a constitutional t(2;3)(q35;q21) was encountered. In addition, analysis of the von Hippel-Lindau gene, VHL, revealed distinct insertion, deletion, and substitution mutations in four of the five tumors tested. On the basis of these results, we conclude that, in this familial case, an alternative route for renal cell carcinoma development is implied. In contrast to the first hit in the generally accepted two-hit tumor-suppressor model proposed by Knudson, the familial translocation in this case may act as a primary oncogenic event leading to (nondisjunctional) loss of the der(3) chromosome harboring the VHL tumor-suppressor gene. The risk of developing renal cell cancer may be correlated directly with the extent of somatic (kidney) mosaicism resulting from this loss.