Chromosome 13q12 region critical for the viability and growth of nasopharyngeal carcinoma hybrids.

Allelic losses of chromosome 13 are often detected in nasopharyngeal carcinoma (NPC) and other cancers, implicating the presence of possible tumor suppressor genes (TSGs) on this chromosome. To identify candidate regions from larger and multiple lost areas observed from direct tumor studies, the technique of monochromosome transfer was utilized to provide functional evidence to verify and define these deletion findings. An intact chromosome 13 was transferred into the NPC HONE1 cell line. Resultant hybrids were used to map putative TSG activity. A critical region at 13q12 was non-randomly eliminated in all surviving microcell hybrids around the marker D13S893; these hybrids were uniformly tumorigenic. Although a known TSG, BRCA2, is mapped close to this critical region, no aberrant expression of this gene was detected in microcell hybrids and other NPC cell lines. These results suggest that at least one novel growth control gene on chromosome 13q12, which is not the BRCA2 gene, is essential for hybrid selection and may play a critical role in tumorigenicity.

Proteasome inhibitors induce growth inhibition and apoptosis in myeloma cell lines and in human bone marrow myeloma cells irrespective of chromosome 13 deletion.

PURPOSE: In this study, we investigated the effects of cell-permeable proteasome inhibitors MG-132, MG-262, PSI, and lactacystin on multiple myeloma cell lines OPM-2, U266, RPMI 8226-S, freshly isolated plasma cells with or without deletion of chromosome 13 from patients with multiple myeloma and plasma cell leukemia, and CD34+ human hematopoietic stem cells. The effects of proteasome inhibitors on cell cycle progression, cell growth, and apoptosis were determined. METHODS: MTT-assay was used to examine the cytotoxicity, and annexin-V staining to quantify apoptosis. Cell cycle analyses were performed using 7-ADD and Ki-67 staining by flow cytometry. RESULTS: PSI was the most potent proteasome inhibitor among those tested with a half maximal cytotoxicity (IC(50)) of 5.7 nM, followed by MG-262, MG-132, and lactacystin. Growth inhibition occurred irrespective of chromosome 13 status. Cell cycle arrest occurred in a dose- and time-dependent manner. Low, subapoptotic dosages led to a partial loss of Ki-67 antigen, whereas apoptotic dosages led to reduced Ki-67 levels. Apoptosis was partially dependent on activation of caspase-3, since Ac-DEVD-cho, a caspase-3 inhibitor, could reduce apoptosis significantly. The cytotoxicity of the four proteasome inhibitors tested was significantly lower in human hematopoietic stem cells than in myeloma cells. CONCLUSIONS: Our results show that proteasome inhibitors induce time- and dose-dependent cell cycle alterations, growth inhibition, and apoptosis in human myeloma cells irrespective of chromosome 13 deletion.

Meiotic behavior of the sex chromosomes in a 45,X/46,X,r(Y)/46,X,dic r(Y) patient whose semen was assessed by fluorescence in situ hybridization.

OBJECTIVE: To determine the meiotic behavior of a ring Y chromosome in a semen sample from a 45,X/46,X,r(Y)/46,X,dic r(Y) patient and the possible interchromosomal effects of the ring on other chromosome pairs. DESIGN: Retrospective analysis. SETTING: Universitat Autònoma de Barcelona. PATIENT: An oligoasthenoteratozoospermic patient who presented for infertility consultation. MAIN OUTCOME MEASURE(S): The sex chromosome content of spermatogenic cells, meiotic figures, and spermatozoa in the ejaculate and the possible interchromosomal effects on chromosomes 13, 18, and 21 were analyzed by using multicolor fluorescence in situ hybridization. Germ-cell aneuploidies were scored. RESULT(S): X0 cells are meiotically incompetent. All meiotic figures were exclusively XY, and 80% showed unpaired sex chromosomes. A high proportion of postreductional cells were XY (45.5%) or nullisomic for sex chromosomes (13.92%). This percentage decreased in spermatozoa to 14.89% and 27.66%, respectively. A statistically significant increase in X-bearing versus Y-bearing cells both in postreductional cells (23.9% vs. 14.3%) and spermatozoa (41.9% vs. 19.3%) was also observed. Evidence for an interchromosomal effect on chromosome 21 was detected. CONCLUSION(S): Data suggest that this patient had a generalized increase incidence of chromosome anomalies, underscoring the importance of incorporating screening for sperm aneuploidies in genetic analysis of affected patients.

p21WAF-1 reorganizes the nucleus in tumor suppression.

Interphasic nuclear organization has a key function in genome biology. We demonstrate that p21WAF-1, by influencing gene expression and inducing chromosomal repositioning in tumor suppression, plays a major role as a nuclear organizer. Transfection of U937 tumor cells with p21WAF-1 resulted in expression of the HUMSIAH (human seven in absentia homologue), Rb, and Rbr-2 genes and strong suppression of the malignant phenotype. p21(WAF-1) drastically modified the compartmentalization of the nuclear genome. DNase I genome exposure and fluorescence in situ hybridization show, respectively, a displacement of the sensitive sites to the periphery of the nucleus and repositioning of chromosomes 13, 16, 17, and 21. These findings, addressing nuclear architecture modulations, provide potentially significant perspectives for the understanding of tumor suppression.

Loss of heterozygosity on chromosome 13 is common only in the biologically more aggressive subtypes of ovarian epithelial tumors and is associated with normal retinoblastoma gene expression.

We examined the frequencies of loss of heterozygosity on chromosome 13 in 77 primary ovarian epithelial tumors subdivided into cystadenomas, tumors of low malignant potential, low grade carcinomas, and high grade carcinomas. Such losses were found in approximately 50% of high grade carcinomas but were not detected in any of the other tumor subtypes (P < 0.0001), suggesting a strong association between these abnormalities and the high grade carcinoma phenotype. The tumors were also examined for abnormalities in expression of the retinoblastoma susceptibility gene (RB). This was assessed by immunohistochemical staining of archival tumor sections with a polyclonal antibody directed against both the phosphorylated and the underphosphorylated forms of the RB protein. Most of the tumors, including those with allelic deletions on chromosome 13, showed normal RB nuclear protein staining patterns. We conclude that loss of RB expression is not a frequent event in primary ovarian carcinomas and that this gene is probably not the target of the frequent allelic deletions on chromosome 13 found in high grade ovarian carcinomas.

Síndrome de Turner 45,XO asociado con una inversión pericéntrica del cromosoma 13 / Turner's syndrome 45, XO associated with a pericentric inversion of chromosome 13

El propósito del presente trabajo es el de describir a una paciente con manifestaciones clínicas del síndrome de Turner, quien al realizarle los estudios cromosómicos en cultivo de linfocitos de sangre periférica, y con técnicas de bandas G, mostró un complemento cromosómico de 45, XO y además una inversión pericéntrica del cromosoma 13 con sus puntos de ruptura en las bandas pll y ql4. Los padres y el hermano de la propósita presentaron un cariotipo normal. Se discuten los mecanismos probables de origen de ambas anomalías y los pocos casos reportados en la literatura

Alteration in the retinoblastoma gene associated with immortalization of human fibroblasts treated with 60Co gamma rays.

Genetic analysis was carried out in human fibroblasts (KMST-6) immortalized by treatment with 60Co gamma rays in order to determine if any genetic change was involved in the immortal transformation of human cells. Analysis by restriction fragment length polymorphism revealed an alteration in chromosome 13q12-14, in which the retinoblastoma (RB) gene locus (13q14) is located. Then the RB gene itself was examined. Structural abnormalities in the RB gene were detected by Southern blot analysis. Furthermore, abnormal RB protein (pRB) was expressed in immortalized KMST-6 cells, as shown by in vitro phosphorylation, whereas normal KMS-6 cells expressed the intact pRB. These findings indicated that inactivation of the RB gene is one of the key events of the immortalization of human cells.

Changes in growth and tumorigenicity following reconstitution of retinoblastoma gene function in various human cancer cell types by microcell transfer of chromosome 13.

Functional loss of the retinoblastoma (RB) gene has been implicated in the initiation or progression of several human tumor types including cancer of the eye, bone, bladder, and prostate. To examine the consequence of adding one RB allele containing its normal regulatory elements back into representative examples of each of these cancer types, as well as to compare the results to those previously reported using various RB complementary DNA constructs, a neomycin resistant marked 13 chromosome was transferred by microcell fusion. Several attempts to obtain RB positive osteosarcoma cells failed. In addition, only one RB positive retinoblastoma clone was isolated. This clone contained many large cells, could not be maintained in long-term culture, and produced only RB negative tumors. Three RB positive bladder cancer cell clones were obtained, all of which grew slower in culture than their RB negative parental counterpart and did not form colonies in soft agar. Tumorigenicity was markedly suppressed in these clones. One clone yielded no tumors, and the other 2 clones produced only one small tumor each, both of which were RB negative. In contrast, the 2 RB positive prostate cancer cell clones isolated had no differences in their cell culture growth properties, including growth in soft agar compared to the parental cells. One of the clones was nontumorigenic, while the other clone produced 4 small tumors, all of which were RB positive. These results indicate that the transfer of one RB allele by microcell transfer produces different levels of growth inhibition as well as tumor suppression, depending on the cell type examined. In the case of prostate cancer, the function of the RB gene in tumor suppression appears to be independent from its growth regulatory function, since no growth inhibition in cell culture was noted in these cells, although tumor suppression was significant.

Precise localization of the excision repair gene, ERCC5, to human chromosome 13q32.3-q33.1 by direct R-banding fluorescence in situ hybridization.

The genomic DNA fragment encoding the excision repair gene, ERCC5, was mapped by direct R-banding fluorescence in situ hybridization. The signals were localized to human chromosome 13q32.3-q33.1. This result was in agreement with previous reports, and the gene was assigned to a narrower region.

Identification of crossovers in Wilson disease families as reference points for a genetic localization of the gene.

Wilson disease (WD) is an autosomal recessive disorder of copper metabolism. A minimum recombinant analysis using D13S22, ESD, RB1, D13S31, D13S55, D13S26, D13S39, and D13S12, all localized at 13q14-q22, has been carried out in 20 WD families of Northwest-European origin. No inconsistencies have been observed with respect to locus order or location of the WD locus (WND) compared with previous linkage studies. D13S31 was mapped as the closest marker proximal to WND, whereas D13S55 and D13S26 were mapped as the closest markers distal to WND. We have identified a crossover between WND and D13S31 in one family and a crossover between WND and D13S55 in another. These crossover sites can be used as reference points for new chromosome 13q14-q21 markers, and are therefore important for a more accurate mapping of the WD locus.

Molecular genetic studies of tumor suppressor gene regions on chromosomes 13 and 17 in colorectal tumors.

BACKGROUND: In the majority of colorectal carcinomas, both copies of the tumor suppressor gene TP53 (tumor protein 53) are known to be inactivated. In contrast to a loss of tumor suppressor function, it has been suggested that an increased copy number of the RB1 gene is involved in the progression of these tumors. PURPOSE: To determine genetic alterations at chromosomes 13 and 17 in colorectal tumors, we have studied several loci on these chromosomes, with special focus on the RB1 and TP53 genes at both the level of DNA sequence and the level of gene expression. METHODS: Restriction fragment length polymorphism analysis was performed after alkaline Southern blotting of the DNA fragments and hybridization (in 7% sodium dodecyl sulfate and 0.5 M NaPO4) of the nylon membranes with multiprimed, radioactively labeled probes. Total RNA was extracted from tissue biopsy specimens by homogenization of the samples in guanidinium thiocyanate followed by separation in a CsCl gradient. By use of an image-processing system, x-ray film signals were measured densitometrically. Point mutations within the TP53 gene were detected by use of polymerase chain reaction (PCR) in combination with constant denaturant gel electrophoresis. Direct sequencing of PCR products revealed the exact nature of the mutations. Protein expression of TP53 was seen by immunostaining of sections from paraffin-embedded material using a mouse monoclonal antibody. The two-sided Fisher's Exact Test was used for statistical analysis. RESULTS: An increase in allelic copy number at 13q loci was seen in 10 (32%) of 31 tumors. In the majority of the cases, this increase probably reflected a change in the diploid status of chromosome 13; in some cases, however, only part of the 13q seemed to be involved. The RB1 gene showed an elevated level of RNA compared with the beta-actin signal. Fourteen (48%) of 29 tumors showed loss of heterozygosity at loci on 17p, and base mutations within the TP53 gene were seen in 14 (42%) of 33 tumors. RNA and protein analyses of TP53 revealed an increased level of expression in the tumors compared with normal mucosa. Allelic variations seen at 13q and 17p were not associated (P = .7). CONCLUSIONS: Our results suggest that, in addition to aneuploidy, gain of specific chromosome 13 sequences is involved in the tumorigenesis of the colon and rectum. In addition, they confirm the importance of TP53 mutations for the progression of such tumors and support the view that accumulation of events is more important than the order of events. The genetic changes observed at chromosome arms 13q and 17p seem to be independent of each other.

Inactivation of the retinoblastoma susceptibility gene in a human high grade non-Hodgkin's lymphoma cell line.

We studied the expression of the retinoblastoma (RB) gene product (p105) in a B-cell line established from a patient with non-Hodgkin's lymphoma (large cell type). The karyotype of this cell line, named Ri-1, showed amongst other changes an apparent deletion of one chromosome 13 on band q14. No p105 could be detected by immunoprecipitation analysis and Western blotting in Ri-1 cells. Northern blotting revealed that RB mRNA is not expressed in Ri-1. Southern blotting confirmed the loss of one RB allele but showed a normal gross structure of the remaining allele. This suggests that the inactivation of the RB gene in Ri-1 cells is due to deletion of one allele and point mutations or small deletions in the other, as is often the case in retinoblastomas. Our findings imply that inactivation of the RB gene may play a role in the pathogenesis of high grade malignant lymphomas and that studies of RB in primary lymphoma samples would be of interest.

Nonrandom chromosomal abnormalities in primary uveal melanoma.

We report on 14 cases of clonal chromosomal anomalies in patients with primary uveal melanoma. An increased dosage of chromosome 8 or of parts of the long arm of chromosome 8 (8q) were detected in eight patients (57%). The smallest multiplied area of 8q appeared to be the region 8q2.1----qter. Monosomy of chromosome 3 was seen in six patients (43%), five of which were associated with anomalies of chromosome 8. Increased dosage of parts of chromosome 8q and loss of heterozygosity of chromosome 3, or the combination of both, seemed to be nonrandom for uveal melanoma and may distinguish it genetically from cutaneous malignant melanoma. Anomalies of chromosome 6, mostly resulting in additional material of 6p or a deletion of 6q, were found in six patients (43%). These anomalies, which seem to be common features of cutaneous malignant melanoma, were considered secondary rather than primary changes in uveal melanoma, since they were present only in subclones in most cases. Loss of the Y chromosome, restricted to tumor cells, was detected in four male patients, and loss of one X chromosome was detected in a female patient.