Mapping of a breast cancer tumor suppressor gene locus to a 4-cM interval on chromosome 18q21

DPC4 and DCC, putative tumor suppressor genes implicated in the genesis of several types of human cancer, lie on the long arm of human chromosome 18. We examined 200 primary breast cancers for allelic losses on chromosome 18, using 15 microsatellite markers distributed along the long arm. Allelic loss was detected most frequently (29-30%) at loci mapped to 18q21. Deletion mapping of the 34 tumors showing partial or interstitial deletions identified a commonly deleted region within the 4-cM interval flanked by D18S474 and D18S487 at 18q21.1-q21.3. Although this interval included the DPC4 and DCC genes, we excluded DPC4 from candidacy when polymerase chain reaction-single-strand conformation polymorphism analysis of each exon failed to detect abnormalities in any of the 54 breast cancers that exhibited loss of heterozygosity involving 18q. Allelic loss on 18q was found more frequently in tumors of the solid tubular histological type (24 of 55, 44%) than in other types (24 of 113, 21%) (P = 0.0049). The results suggest that a tumor suppressor gene located within the 4-cM region at 18q21, either DCC or another gene not yet identified, may play a role in the development of some sporadic breast cancers, particularly those of the solid tubular type.     

Hyperparathyroidism-jaw tumor syndrome: the HRPT2 locus is within a 0.7-cM region on chromosome 1q

Hyperparathyroidism-jaw tumor syndrome (HPT-JT) is an autosomal dominant disease characterized by the development of multiple parathyroid adenomas and multiple fibro-osseous tumors of the maxilla and mandible. Some families have had affected members with involvement of the kidneys, variously reported as Wilms tumors, nephroblastomas, and hamartomas. The HPT-JT gene (HRPT2) maps to chromosome 1q25-q31. We describe further investigation of two HPT-JT families (K3304 and K3349) identified through the literature. These two expanded families and two previously reported families were investigated jointly for linkage with 21 new, closely linked markers. Multipoint linkage analysis resulted in a maximum LOD score of 7.83 (at recombination fraction 0) for markers D1S2848-D1S191. Recombination events in these families reduced the HRPT2 region to approximately 14.7 cM. In addition, two of these four study families (i.e., K3304 and K11687) share a 2.2-cM length of their (expanded) affected haplotype, indicating a possible common origin. Combining the linkage data and shared-haplotype data, we propose a 0.7-cM candidate region for HRPT2.     

Localization of a novel tumor suppressor gene associated with human oral cancer on chromosome 4q25

Recent cytogenetic and molecular studies with highly polymorphic microsatellite markers have implicated allele loss involving chromosome 4 in several human cancers, which suggests the presence of multiple tumor suppressor gene (TSG) loci. However, there has been no detailed analysis of loss of heterozygosity (LOH) on chromosome 4 in oral squamous cell carcinoma (OSCC). To determine the location of a putative TSG associated with OSCC on chromosome 4, polymerase chain reaction (PCR) analysis of microsatellite polymorphisms corresponding to 17 loci was performed to screen 32 patients with OSCC. LOH was observed in the majority of the tumors (75%) in at least one of the loci. The loci on the long arm exhibited a significantly higher frequency of deletions (66%) than those of the short arm (25%). Among the loci tested, frequent LOH was centered at D4S1573 on 4q25, which represents a region of about 4 centimorgans (cM). However, no commonly deleted regions were found on the short arm of the chromosome. We detected microsatellite instability (MI) in 31% of the cases. MI was also observed more frequently on the long arm (28%) than the short arm (6%). Thus, our data indicate that alterations of chromosome 4 regions, especially the long arm, are associated with OSCC tumorigenesis and that the 4q25 region may harbor at least one putative TSG.     

Deletion mapping of two potential chromosome 14 tumor suppressor gene loci in ovarian carcinoma

Previous allelotyping studies of epithelial ovarian carcinoma suggest that loss of heterozygosity on chromosome 14q may be a common genetic alteration in this tumor type. The purpose of this study was to determine a precise frequency of chromosome 14q allelic loss in ovarian carcinomas and to define a minimal region(s) of deletion. Seventy-six ovarian carcinomas representative of the complete spectrum of grade, stage, and histological subtype were selected for PCR-based deletion mapping analysis using 15 highly polymorphic microsatellite markers spanning the length of this chromosome arm. Loss of heterozygosity was observed in 49% of the tumors studied, placing 14q among the most frequently affected chromosomal regions in ovarian cancer. Deletions were observed in all tumor grades and stages and in all histological subtypes except tumors of low malignant potential. Deletion of the entire chromosome arm was rare; the majority of tumors displayed partial losses, providing an informative basis for detailed deletion mapping. Two distinct minimal regions of deletion were delineated. One region was defined by markers D14S80 and D14S75 at 14q12-13, and the other region was defined by markers D14S65 and D14S267 at 14q32. These data implicate the involvement of two tumor suppressor genes on chromosome 14q in a substantial fraction of ovarian carcinomas.     

Pten, a candidate tumor suppressor gene, maps to mouse chromosome 19

One hundred and five sequential transjugular core liver biopsies (TJLBx) were performed in 101 patients with coagulopathy and/or ascites using the 19-gauge Quick-Core Biopsy (QCB) needle. Two-hundred and seventy-three cores were obtained in 295 passes (92. 5%). One-hundred and two of the 105 procedures (97.1%) led to a histopathologic diagnosis. One of the three nondiagnostic biopsies was done because of severe autolysis of the liver. There was one subcapsular hematoma, one hepatic arteriovenous fistula, and one liver capsular puncture. Two minor neck hematomas occurred. One death was reported (unrelated to the procedure). QCB needle TJLBx is an effective and relatively safe way to obtain core liver samples.     

Cytogenetic localization of genetic markers on porcine chromosome 7q

We report two cases of port site metastasis as the presenting feature of colonic and ovarian carcinoma after laparoscopic cholecystectomy. Cholecystectomy was performed for upper abdominal pain and gallstones. Six and 4 months after the operation the patients presented with nodules at port sites, other than the site of extraction. Biopsy proved both to be adenocarcinoma and further management found these to be from advanced caecal and ovarian carcinomas.     

Allelic loss on chromosome 22 in oral cancer: possibility of the existence of a tumor suppressor gene on 22q13

PURPOSE: To model the influence of hypoxic radioprotection in fractionated treatments over a range of fraction sizes. To determine whether there is a "therapeutic window" of dose per fraction where hypoxic radioresistance could be reduced, and if so, where it occurs in different cell lines. MATERIALS AND METHODS: A mathematical model has been used to simulate the response of cells to low doses of radiation, in the region of clinical interest. We have used the inducible repair variant of the linear quadratic (LQ) equation, with a hypersensitive region (alphaS) at low doses that gradually transforms to the accepted "resistance" in the shoulder region (alphaR). It contains two new parameters, the ratio alphaS/alphaR, and D(C). We have accepted that the "induction dose" D(C) is modified by anoxia to the same extent as the other parameters. We have initially modeled using theoretical parameters and then checked the conclusions with 14 sets of published experimental data for cell lines investigated for inducible repair. RESULTS: We have computed the clinical hypoxic protection (OER') as a function of dose per fraction in simulations of clinical fractionated schedules. We have identified a therapeutic window in terms of dose per fraction at about 0.5 Gy, where the OER' is minimized, regardless of the precise cell survival curve parameters. The minimum OER' varies from one cell line to another, falling to about 1.0 if alphaS/alphaR = 6-10 and even far below 1.0 if alphaS/alphaR > or = 20. DISCUSSION: Hyperfractionation using 0.5 Gy fractions may therefore be more effective than oxygen mimetic chemical sensitizers, since it could even make some tumor cells more sensitive than oxic normal tissues. The tumor lines that benefit most from this type of sensitization are those with the highest intrinsic oxic radioresistance, i.e. those with high SF2 values.     

The murine CYLN2 gene: genomic organization, chromosome localization, and comparison to the human gene that is located within the 7q11.23 Williams syndrome critical region

Several agents that act through G-protein-coupled receptors and also stimulate phosphoinositide-specific phospholipase C (PI-PLC), including angiotensin II, vasopressin, norepinephrine, and prostaglandin (PG) F2alpha, activated the ERK1 (p44mapk) and ERK2 (p42mapk) members of the mitogen-activated protein (MAP) kinase family in primary cultures of rat hepatocytes, measured as phosphorylation of myelin basic protein (MBP) by a partially purified enzyme, immunoblotting, and in-gel assays. All these agonists induced a peak activation (two to threefold increase in MBP-phosphorylation) at 3-5 min, followed by a brief decrease, and then a sustained elevation or a second increase of the MAP kinase activity that lasted for several hours. Although all the above agents also stimulated PI-PLC, implicating a Gq-dependent pathway, the elevations of the concentration of inositol (1,4,5)-trisphosphate did not correlate well with the MAP kinase activity. Furthermore, pretreatment of the cells with pertussis toxin markedly reduced the MAP kinase activation by angiotensin II, vasopressin, norepinephrine, or PGF2alpha. In addition, hepatocytes pretreated with pertussis toxin showed a diminished MAP kinase response to epidermal growth factor (EGF). The results indicate that agonists acting via G-protein-coupled receptors have the ability to induce sustained activation of MAP kinase in hepatocytes, and suggest that Gi-dependent mechanisms are required for full activation of the MAP kinase signal transduction pathway by G-protein-coupled receptors as well as the EGF receptor.     

Localization of the gene for congenital dyserythropoietic anemia type I to a <1-cM interval on chromosome 15q15.1-15.3

Congenital dyserythropoietic anemias (CDA) are a rare group of red-blood-cell disorders of unknown etiology that are characterized by ineffective erythropoiesis, pathognomonic cytopathology of the nucleated red blood cells in the bone marrow, and secondary hemochromatosis. In CDA type I, bone-marrow electron microscopy reveals characteristic findings in erythroid precursors, including spongy heterochromatin and enlarged nuclear pores. Since the genetic basis of CDA type I is not evident, we used homozygosity and linkage mapping to localize the genetic defect responsible for CDA type I in 25 Bedouins from four large consanguineous families. We report the linkage of this disease to markers on chromosome 15 located at q15. 1-q15.3. Fourteen markers within a 12-cM interval were typed in the relevant family members. Nine of the markers yielded maximum LOD scores of 1.625-12.928 at a recombination fraction of .00. Linkage disequilibrium was found only with marker D15S779. Haplotype analysis revealed eight different carrier haplotypes and highlighted the existence of a founder haplotype. Identification of historical crossover events further narrowed the gene location to between D15S779 and D15S778. The data suggest localization of the CDA type I gene within a 0.5-cM interval. The founder mutation probably occurred >/= 400 years ago. Sequence analysis of the coding region of protein 4.2, the only known erythroid-specific gene in the locus, did not reveal any change in the CDA type I patients. Future analysis of this locus may lead to the identification of a gene essential to normal erythropoiesis.     

P-TEN, the tumor suppressor from human chromosome 10q23, is a dual-specificity phosphatase

Protein tyrosine phosphatases (PTPs) have long been thought to play a role in tumor suppression due to their ability to antagonize the growth promoting protein tyrosine kinases. Recently, a candidate tumor suppressor from 10q23, termed P-TEN, was isolated, and sequence homology was demonstrated with members of the PTP family, as well as the cytoskeletal protein tensin. Here we show that recombinant P-TEN dephosphorylated protein and peptide substrates phosphorylated on serine, threonine, and tyrosine residues, indicating that P-TEN is a dual-specificity phosphatase. In addition, P-TEN exhibited a high degree of substrate specificity, showing selectivity for extremely acidic substrates in vitro. Furthermore, we demonstrate that mutations in P-TEN, identified from primary tumors, tumor cells lines, and a patient with Bannayan-Zonana syndrome, resulted in the ablation of phosphatase activity, demonstrating that enzymatic activity of P-TEN is necessary for its ability to function as a tumor suppressor.     

Homozygosity and linkage-disequilibrium mapping of the urofacial (Ochoa) syndrome gene to a 1-cM interval on chromosome 10q23-q24

The urofacial (Ochoa) syndrome (UFS) is a rare autosomal recessive disease characterized by congenital obstructive uropathy and abnormal facial expression. The patients present with enuresis, urinary-tract infection, hydronephrosis, and voiding dysfunctions as a result of neurogenic bladders. To map the UFS gene, a genome screen using a combination of homozygosity-mapping and DNA-pooling strategies was performed in 20 selected patients, one patient pool, and three control pools (unaffected relatives). After analyses of 36 randomly chosen markers, D10S677 was identified as being linked to and associated with UFS, as suggested by a significant excess of homozygosity in patients compared with that in unaffected relatives (P < 10(-6)), as well as by the allelic-frequency differences between the patient pool and control pools. Ten additional markers flanking D10S677 and covering a 22-cM region then were analyzed to fine-map the UFS gene by use of haplotype (linkage disequilibrium) analysis. All 31 patients were found to be homozygous for two closely linked markers (D10S1726 and D10S198) located approximately 5 cM telomeric to D10S677, whereas only 12% of the unaffected relatives were homozygous for both markers (P < 10(-19)). Several patients are heterozygous at two markers immediately flanking D10S1726/D10S198, one on the centromeric side (D10S1433) and the other on the telomeric side (D10S603). These recombinational events place the UFS gene near D10S1726/D10S198 and within a 1-cM interval defined by D10S1433 and D10S603 on chromosome 10q23-q24.     

Refined localization of the asparagine synthetase gene (ASNS) to chromosome 7, region q21.3, and characterization of the somatic cell hybrid line 4AF/106/KO15

We have mapped the asparagine synthetase gene (ASNS) to 7q21.3 by fluorescence in situ hybridization. While this study refined the localization of the gene, it also revealed a rearrangement in a somatic cell hybrid line which was used in previous ASNS mapping. Using additional probes from other regions of human chromosome 7, we showed that this cell line (4AF/106/KO15) contained a rearranged chromosome 7 in which a segment of the long arm was apparently duplicated and inserted into the short arm. Caution should be used therefore when interpreting data obtained from this cell line for gene mapping studies.     

Localization and characterization of a chromosome 11 tumor suppressor gene using organotypic raft cultures

The development and progression of human cancer often involves the inactivation of tumor suppressor gene function. Alterations in human chromosome 11 during the development of human cutaneous squamous cell carcinoma suggest the presence of a tumor suppressor gene on this chromosome. Moreover, previous studies in our laboratory demonstrated the presence of a functional tumor suppressor gene on chromosome 11 for the human cutaneous squamous cell carcinoma cell line A388.6TG.c2. In this investigation, we have used organotypic culturing of epithelial cells as a novel in vitro assay for tumor suppression. A388.6TG.c2 and control cells form an abnormal stratified epithelium of 8-12 layers when cultivated on organotypic rafts. In contrast, the chromosome 11 microcell hybrids, HMC 100p4B and HMC 100p5A, form an epithelium of only two to three cell layers. This in vitro growth suppression of the chromosome 11 microcell hybrids in the organotypic rafts correlates well with our previous in vivo skin graft experiments. Comparison of the proliferation and apoptotic indices of cell lines grown on the organotypic rafts suggests that the tumor suppressor gene on chromosome 11 has restricted the ability of the microcell hybrids to stratify but has not significantly altered their ability to undergo cell division or programmed cell death. Furthermore, flow cytometric analysis of cells grown on organotypic raft cultures suggests that the chromosome 11 microcell hybrids are actively progressing through the cell cycle rather than arrested in a particular stage. We have used this novel application of organotypic raft cultures to further localize the chromosome 11 tumor suppressor gene. Introduction of a single der(11)t(X;11) chromosome lacking most of the long arm of chromosome 11 into A388.6TG.c2 does not affect growth on organotypic raft cultures. These data suggest the tumor suppressor gene maps to the long arm of chromosome 11 in the region of 11q13-qter.     

Refined chromosomal localization of the putative tumor suppressor gene TP73

We report the identification of a mouse cDNA Tpd52l1 (tumor protein D52-like 1), which represents the first demonstrated orthologue of the human TPD52L1 (alias D53) gene, a member of the breast carcinoma-associated TPD52 (alias D52) gene family. In situ hybridization mapping located the Tpd52l1 gene to chromosome 10A4-10B2. Since the TPD52L1 gene is found at human chromosome 6q22-->q23, the mouse and human TPD52L1 loci are syntenically conserved.     

The putative tumor suppressor gene on chromosome 5q for hepatocellular carcinoma is distinct from the MCC and APC genes

We have previously shown that the tumor suppressor gene for hepatocellular carcinoma (HCC) without cirrhosis may be located on chromosome 5q35-qter. In this study, we analyzed nine cases of primary HCC without cirrhosis using probes from the MCC and APC genes, which are in the region 5q21-22. None of the informative cases had allele loss detected by these probes, whereas the probe lambda MS8 for the region 5q35-qter showed allele loss in six out of six informative cases. The results confirm that the putative tumor suppressor gene for HCC without cirrhosis on chromosome 5q is distinct from the MCC and APC genes.     

Localization of tumor suppressor activity important in nonsmall cell lung carcinoma on chromosome 11q

Loss of heterozygosity on chromosome 11q23 is observed at high frequency in human nonsmall cell lung carcinomas (NSCLCs), suggesting the presence of a tumor suppressor gene. Previous analysis of DNA from 79 patients identified a commonly deleted segment of 5 centimorgans. Complementation analysis was used to further localize a putative tumor suppressor gene. Three yeast artificial chromosome (YAC) clones spanning the minimal loss of heterozygosity region were modified, and spheroplast fusion was used to transfer them into human A549 NSCLC or murine Lewis lung carcinoma (LLC) cell lines. The resulting yeast x human hybrid cell lines containing an intact copy of a 1.6-Mb YAC, 939b12, showed reduced growth in vitro. Injection of parental A549 cells into athymic (nu/nu) mice resulted in tumor formation at 27 of 28 injection sites. In contrast, two independent 939b12-containing cell lines formed tumors at only 3 of 20 injection sites. 939b12 also suppressed tumor formation by LLC NSCLC cells in nude mice, but YACs 785e12 and 911f2, which flank 939b12, had no suppressor activity. Further localization of tumor suppression activity on 939b12 was accomplished by introduction of defined fragmentation derivatives into A549 cells and by analysis of YACs that were broken on transfer into LLC cells. This complementation approach localized tumor suppression activity to the central 700 kb of 939b12 and provides a functional assay for positional cloning of this tumor suppressor gene.     

Regional localization of human M-BCR gene to chromosome 23 band q11 in the great apes

Cassettes based on a hisG-URA3-hisG insert have been modified by the addition of an KmR-encoding gene and flanking polylinker sites, greatly simplifying construction of gene disruption vectors in Escherichia coli. After gene disruption in yeast, URA3 can then be excised by recombination between the hisG repeats flanking the gene, permitting reuse of the URA3 marker.     

A novel tumor suppressor locus on chromosome 18q involved in the development of human lung cancer

The high incidence of loss of heterozygosity (LOH) on chromosome 18q in advanced non-small cell lung carcinomas indicates the presence of tumor suppressor gene(s) on this chromosome arm, which plays an important role in the acquisition of malignant phenotypes in lung cancers. In the present study, we examined 62 lung cancer specimens and 54 lung cancer cell lines for allelic imbalance at 11 microsatellite loci to define common regions of 18q deletions. Allelic imbalance of 18q was detected in 24 (55.8%) non-small cell lung carcinoma specimens and in 6 (31.6%) small cell lung carcinoma specimens, whereas a similar frequency of LOH was statistically inferred to occur in cell lines by analyzing marker homozygosity as an indirect measure of LOH. Five specimens and 11 cell lines showed partial or interstitial deletions of chromosome 18q, and 2 of them had homozygous deletions at the 18q21.1 region. A commonly deleted region was assigned between the D18S46 and y953G12R loci. The size of this region is less than 1 Mb, and the coding exons of three candidate tumor suppressor genes, Smad2, Smad4, and DCC, were mapped outside the region. This result suggests that the common region harbors a novel tumor suppressor gene involved in the progression of lung cancer.     

Mapping of rat bilirubin UDP-glucuronosyl-transferase gene (Ugt1a1) to chromosome region 9q35-->q36

Bilirubin and phenol UDP-glucuronosyltransferases (UGTs) are located on the same chromosome and comprise the UGT1 gene complex. A 1,763-bp cDNA probe (UGT1*0) specific for rat liver bilirubin UGT was used to localize the UGT1 complex locus (Ugt1a1) to chromosome region 9q35-->q36 by fluorescence in situ hybridization. This assignment is the first report on the location of a gene of the rat UGT1 complex using high-resolution banded metaphase chromosomes.