Methylation of death-associated protein kinase in ovarian carcinomas.

Death-associated protein (DAP) kinase is a serine/threonine kinase that plays an integral role in apoptosis and metastasis. The purpose of our study was to determine the methylation status of DAP kinase in ovarian carcinomas. Thirty-one patients with histologically confirmed epithelial ovarian cancers treated at Roswell Park Cancer Institute, Buffalo, New York, between 1987 and 1999 were studied. Sixty-two samples were examined for DAP kinase methylation status: 1 normal human genomic DNA sample from a healthy individual, 1 transformed normal surface ovarian epithelial cell line (IOSE, from Dr Nancy Auersperg, Vancouver, Canada), 2 ovarian carcinoma cell lines (OVCAR3 and A2780), 1 ovarian serous cystadenoma, and 30 ovarian carcinomas. Additionally, peripheral blood DNA was examined from the patients with the serous cystadenoma and ovarian carcinomas. Methylation-specific polymerase chain reaction was performed using primers designed for the unmethylated and methylated promoter regions. The DAP kinase gene was unmethylated in both the normal human genomic DNA sample and the transformed normal surface epithelial ovarian cell line. The two ovarian cancer cell lines were methylated. In the 30 patients with malignant disease, methylation of DAP kinase was observed in 20 (67%). Peripheral blood DNA was available in 26 (87%) of the 30 patients. Comparison of the paired samples indicated that 14 (54%) were methylated and 12 (46%) were unmethylated. There was no correlation between the DAP kinase methylation status and stage, grade, histology, or survival. Methylation of CpG islands in the promoter region of the DAP kinase gene is common in peripheral blood DNA and tissue samples of patients with ovarian carcinomas. This molecular aberration may represent a potential target for therapeutic intervention.

Genome-wide allelotyping indicates increased loss of heterozygosity on 9p and 14q in early age of onset colorectal cancer.

Colorectal cancer remains a significant public health challenge, despite our increased understanding of the genetic mechanisms involved in the initiation and progression of this disorder. It has become clear that multiple mechanisms lead to the tumorigenic phenotype, with familial predisposition syndromes accounting for less than 15% of all colorectal cancers. A genome-wide scan for loss of heterozygosity (LOH) was carried out with 150 highly polymorphic markers in an effort to identify additional loci involved in colorectal tumorigenesis in DNA samples from 42 colorectal cancer patients. The results confirm earlier observations that tumor DNAs from patients with hereditary nonpolyposis colon cancer (HNPCC) either maintain heterozygosity or exhibit altered or additional alleles. DNAs from patients with early onset colorectal carcinomas (diagnosed prior to age 50) revealed a higher overall degree of LOH than DNAs from patients with sporadic colorectal cancers diagnosed later in life (after age 50). While regions on 1p, 10q and 14q are suggestive, statistical analysis of LOH at these regions failed to reach significance. However, LOH at 9p did reveal a statistically significant increase in the early onset patient group, compared to the greater than age 50 group. LOH on 9p may involve inactivation of p16/CDKN2 through aberrant DNA methylation on the remaining chromosome, resulting in a situation analogous to a homozygous deletion of p16 and providing a selective growth advantage to these cells. This marker may prove to be a useful prognostic indicator for patient stratification in the design of therapy for early onset colorectal cancer patients.

Novel hMLH1 and hMSH2 germline mutations in African Americans with colorectal cancer.

CONTEXT: Germline mutations of the DNA mismatch repair (MMR) genes hMLH1 and hMSH2 have been shown to cosegregate with the colorectal cancer phenotype in multiple hereditary nonpolyposis colorectal cancer (HNPCC) pedigrees. However, the frequency of these mutations among African American patients with colorectal cancer is unknown. OBJECTIVE: To investigate the frequency of germline alterations of the DNA MMR genes hMLH1 and hMSH2 among African Americans affected by HNPCC and early-age onset colorectal cancer. DESIGN, SETTING, AND PATIENTS: Forty unrelated African American HNPCC and early-age onset colorectal cancer patients (8 women, 3 men) were identified from the cancer registry at a National Cancer Institute-designated referral center, 11 of whom were available for and agreed to study participation from January 1997 to February 1998. The mean age of the subjects was 44 years. An additional 50 age- and sex-matched African Americans without personal or family history of colorectal, endometrial, ovarian, urinary tract, or upper gastrointestinal tract malignancy were also studied as a polymorphism control population. In all subjects, genomic DNA was amplified by polymerase chain reaction for all hMLH1 and hMSH2 exons and screened using single-strand conformation polymorphism (SSCP) analysis. Samples demonstrating significant SSCP shifts underwent automated nucleotide sequencing analysis. MAIN OUTCOME MEASURE: Frequency of hMLH1 and hMSH2 germline alterations in the affected and control subjects. RESULTS: Germline hMLH1 and hMSH2 mutations were detected in 3 (27%) of the African American colorectal cancer probands studied. Each mutation was novel. Two hMLH1 (an A-->T transversion at codon 26 and a GG-->AT substitution across codons 177 and 178) mutations and 1 hMSH2 mutation (a C-->T transition at codon 389) were identified in 3 female study subjects. Six other hMLH1 and hMSH2 alterations were detected but were presumed to be polymorphisms. Neither missense mutation (at codons 26 and 389) was detected in the control population. CONCLUSIONS: The results of our analysis support an association between the 3 mutations reported and predisposition to colorectal cancer. Further studies are needed to define DNA MMR gene-associated colorectal cancer in African Americans, an understudied population at increased risk of fatal colorectal cancer.

Genomic DNA-based hMSH2 and hMLH1 mutation screening in 32 Eastern United States hereditary nonpolyposis colorectal cancer pedigrees.

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant cancer syndrome characterized by early age of onset colorectal cancer (mean 45 years) as well as endometrial, urinary tract, and upper gastrointestinal adenocarcinomas. The HNPCC phenotype has been shown to segregate with germline mutations in the human homologues of the DNA mismatch repair genes MSH2, MLH1, PMS1, and PMS2. However, the majority of published DNA mismatch repair gene mutation surveys associated with HNPCC kindreds report multiple levels of preselection, including 2p and 3p chromosomal linkage analysis and the evaluation of microsatellite instability of proband colorectal cancers prior to mutation analysis. For this reason, the concise contribution of each of the known DNA mismatch repair genes to the HNPCC phenotype remains unknown. We report the results of a genomic DNA-based analysis of hMSH2 and hMLH1 germline mutations in 32 unrelated Eastern United States HNPCC kindreds. These families were selected for study on the basis of phenotype only. We identified three hMSH2 and six hMLH1 mutations in eight families, for a positive mutation rate of 25%. Two mutations were identified in one of the families. Four of the mutations detected have not been reported in the literature previously. One of the mutation-positive families is African American; the others were of European-American ancestry. These results provide a clarification of the contribution of hMSH2 and hMLH1 to the HNPCC phenotype and suggest that in the majority of Eastern United States HNPCC kindreds selected by phenotype alone, the molecular genetic basis for the disease remains unknown.

Linkage of phosphoribosylpyrophosphate synthetases 1 and 2, Prps1 and Prps2, on the mouse X chromosome.

The X Chromosome (Chr) genes for phosphoribosylpyrophosphate synthetases 1 and 2, Prps1 and Prps2, were mapped on the mouse X Chr with interspecific backcrosses between C57BL/6 (B6) and M. spretus (S). Southern analysis showed that Prps1 mapped between Plp and DXWas31, a mouse X Chr region that is homologous to Xq21-24 on the human X Chr while Prps2 mapped between DXWas31 and Amg, a region that is homologous to the map position of PRPS2 on Xp22 of the human X Chr. Additionally, other restriction fragments highlighted by PRS II showed autosomal segregation. In situ hybridization and FISH analysis of metaphase chromosome spreads prepared from lymphocytes of B6 or S male mice confirmed that there were in fact two different locations on the X Chr, X F1-2 and X F2-3 for Prps1 and 2 respectively, as well as two autosomal sites for Prps-like genes.

Mapping and expression of the ubiquitin-activating enzyme E1 (Ube1) gene in the mouse.

The nucleotide sequence of the human cDNA encoding ubiquitin-activating enzyme E1 is more than 99% identical with the human A1S9T cDNA, a gene that has been shown to complement the temperature-sensitive mutant mouse cell line, tsA1S9. The amino acid sequences of the proteins encoded by these two cDNA sequences are identical, and both cDNAs were previously shown to be located in the same region of the human X chromosome; thus, ubiquitin-activating enzyme E1 and A1S9T appear to be the same gene, designated UBE1. By in situ hybridization to metaphase chromosomes from male mice and by Southern blot analysis of male and female mouse DNA, we show that, in the mouse, a human UBE1 cDNA probe identified both X- and Y-linked loci. Ube1 is located at band A2 of the mouse X Chromosome (Chr) and Ube2 on the short arm of the Y Chr. This is in contrast to the situation in the human, where there is no evidence for Y-linked sequences related to UBE1. Mapping of the Ube1 gene in interspecific backcrosses between Mus spretus and C57BL/6 shows that the Ube1 locus maps close to Timp, in a conserved region of the mouse and human X Chrs that include Otc, Cybb, Syn1, Timp, and Araf. Expression of Ube1 on the inactive X Chr was examined to determine whether this gene is subject to X-Chr inactivation in the mouse, as there is previous evidence that the human UBE1 gene escapes, at least partially, X inactivation. Sequencing of reverse transcriptase polymerase chain reaction (RT-PCR) products from M. spretus, C57BL/6J, and T(X;16)16H x M. spretus F1 female mice indicates that the mouse Ube1 gene is subject to X-Chr inactivation in vivo. This represents a new example of differences between the sex chromosomes of mouse and human.

Linkage of amelogenin (Amel) to the distal portion of the mouse X chromosome.

Amelogenins are hydrophobic, proline-rich proteins that are the primary biosynthetic products of ameloblasts. These cells are responsible for the formation of tooth enamel, and amelogenins play an important role in the process of biomineralization. A cDNA, corresponding to the mouse 26-kDa amelogenin, has been molecularly cloned and sequenced. Southern blot analysis of genomic DNA from the mouse using this cDNA as a probe indicates that there is only one mouse amelogenin (Amel) gene. This paper describes restriction site variation for the Amel gene that we have identified between C57BL/6 and M. spretus and the segregation of that variation as an X-chromosome gene. The position of the amelogenin locus (Amel) relative to the loci for alpha-galactosidase (Ags), proteolipoprotein (Plp), and the random genomic probe DXWas31 has been determined. Amel is established as: (1) the most distal locus in the genetic map of the mouse X chromosome, (2) lying proximal to the X:Y pairing region, and (3) being restricted to the mouse X chromosome.

Linkage of the erythroid transcription factor gene (Gf-1) to the proximal region of the X chromosome of mice.

We have used a cDNA probe for mouse Gf-1 gene that encodes the erythroid cell transcription factor to identify genetic variation in genomic DNA between Mus species. The segregation of Gf-1 DNA variation was analyzed in Mus species crosses that have been previously typed for the segregation of more than 30 genes spanning 80 cM of the mouse X chromosome from the centromere to the border of the X-Y pairing region. We identified a single X chromosome locus in the mouse, Gf-1, and an analysis of recombinants from 203 backcross progeny mapped Gf-1 to the proximal portion of the chromosome, coincident with the Cybb locus and proximal to Otc gene locus. A gene order of centromere, DXWas70, Cybb/Gf-1, Otc, Timp was established for the mouse X chromosome, which is in agreement with the map position observed on the human X chromosome.

Linkage of a gene for neural cell adhesion molecule, L1 (CamL1) to the Rsvp region of the mouse X chromosome.

L1 is a glycoprotein with an apparent molecular weight of 200 kDa in the developing fetus and adult central nervous system. In the peripheral nervous system, it has a molecular weight of 230 kDa. The L1 protein appears to be encoded by a single gene that has been located on the human X chromosome by in situ hybridization. In this paper we describe restriction variation in genomic DNA Southern analysis between Mus species for the K13 cDNA probe for the L1 neural cell adhesion molecule. We have designated the locus described by this variation as cell adhesion molecule L1, CamL1. The X chromosome linkage and the relative position on the X chromosome coincident with the genes Rsvp/G6pd/Cf-8 were defined in backcross matings involving M. spretus and M. musculus.

Localization of the mouse Mcf-2 (Dbl) protooncogene within a conserved linkage group on the mouse X chromosome.

A mouse cDNA probe homologous to the human MCF2 transforming sequence has been identified and partially cloned, and is used here to localize the gene on the mouse X chromosome. The human gene has been physically mapped to within 60 kb of the gene for coagulation factor IX, within a large conserved linkage group between the mouse and human genomes which extends from HPRT to G6PD on the X chromosomes of both mammalian species. In situ hybridization of the mouse Mcf-2 probe onto mouse metaphase chromosomes indicates that this gene lies in the same region of the X chromosome as Cf-9, the mouse gene for coagulation factor IX. Moreover, segregation of species-specific genomic DNA polymorphisms for Mcf-2 and Cf-9 in a total of 203 individuals derived from two large interspecific mouse backcross populations (which are also segregating for 17 other X-linked molecular markers) demonstrates that the mouse genes are separated by only 0.5 +/- 0.5 cM. Despite this short distance we were able to order Mcf-2 and Cf-9 relative to one another and other genes in this region. The mouse gene order Hprt-Cf-9-Mcf-2-G6pd predicts a similar ordering of genes on the human X chromosome, a gene order which has only recently been demonstrated by physical mapping. Thus, the map location and linkage relationships of the Mcf-2 gene are similar in man and mouse, and this unique protooncogenic locus is part of a conserved linkage group on the mammalian X chromosome.

The 5' ends of yeast killer factor RNAs are pppGp.

The 5' nucleotides of the double-stranded RNAs of yeast killer factor have been isolated by digestion with pancreatic, T1 and T2 RNase followed by two-dimensional electrophoresis. They were identified by bacterial alkaline phosphatase and snake venom phosphodiesterase digestions. Both the larger double-stranded RNA (L, of 2.5 x 10(6) daltons) and the smaller double-stranded RNA (M, of 1.4 x 10(6) daltons) have the 5' end groups pppGp. These 5' ends are dissimilar to those of the double-stranded RNAs of animal viruses but may be characteristic of the 5' ends of the double-stranded RNAs of fungal viruses.