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Biomedical subjects

A Kamb

Publications and source records attributed to A Kamb.

At least 19 recordsLinked to original sources
PubMed1996

Reversible, p16-mediated cell cycle arrest as protection from chemotherapy.

A model system has been developed to explore the relationship between cell cycle arrest and chemotherapeutic toxicity. An isopropyl-1-thio-beta-D-galactopyranoside-inducible P16 construct was introduced stably into a melanoma cell line and used to promote G0-G1 arrest in the recipient cells. The state of arrest was reversible and did not compromise cell viability over a period of at least 7 days. Isopropyl-1-thio-beta-D-galactopyranoside-treated, arrested cells were significantly more resistant to the chemotherapeutic agents methotrexate (approximately 50 times), vinblastine (>100 times), and cisplatin (approximately 10 times) compared to controls. This strategy of protection from chemotherapy exploits one of the basic genotypic differences between normal cells and tumor cells: the integrity of genetic pathways that regulate growth.

Antineoplastic Agents↗
PubMed1996

The complete BRCA2 gene and mutations in chromosome 13q-linked kindreds.

Breast carcinoma is the most common malignancy among women in developed countries. Because family history remains the strongest single predictor of breast cancer risk, attention has focused on the role of highly penetrant, dominantly inherited genes in cancer-prone kindreds (1). BRCA1 was localized to chromosome 17 through analysis of a set of high-risk kindreds (2), and then identified four years later by a positional cloning strategy (3). BRCA2 was mapped to chromosomal 13q at about the same time (4). Just fifteen months later, Wooster et al. (5) reported a partial BRCA2 sequence and six mutations predicted to cause truncation of the BRCA2 protein. While these findings provide strong evidence that the identified gene corresponds to BRCA2, only two thirds of the coding sequence and 8 out of 27 exons were isolated and screened; consequently, several questions remained unanswered regarding the nature of BRCA2 and the frequency of mutations in 13q-linked families. We have now determined the complete coding sequence and exonic structure of BRCA2 (GenBank accession #U43746), and examined its pattern of expression. Here, we provide sequences for a set of PCR primers sufficient to screen the entire coding sequence of BRCA2 using genomic DNA. We also report a mutational analysis of BRCA2 in families selected on the basis of linkage analysis and/or the presence of one or more cases of male breast cancer. Together with the specific mutations described previously, our data provide preliminary insight into the BRCA2 mutation profile.

BRCA2 Protein↗
PubMed1996

Low incidence of BRCA2 mutations in breast carcinoma and other cancers.

Inherited mutant alleles of familial tumour suppressor genes predispose individuals to particular types of cancer. In addition to an involvement in inherited susceptibility to cancer, these tumour suppressor genes are targets for somatic mutations in sporadic cancers of the same type found in the familial forms. An exception is BRCA1, which contributes to a significant fraction of familial breast and ovarian cancer, but undergoes mutation at very low rates in sporadic breast and ovarian cancers. This finding suggests that other genes may be the principal targets for somatic mutation in breast carcinoma. A second, recently identified familial breast cancer gene, BRCA2 (refs 5-8), accounts for a proportion of breast cancer roughly equal to BRCA1. Like BRCA1, BRCA2 behaves as a dominantly inherited tumour suppressor gene. Individuals who inherit one mutant allele are at increased risk for breast cancer, and the tumours they develop lose the wild-type allele by heterozygous deletion. The BRCA2 coding sequence is huge, composed of 26 exons that span 10,443 bp. Here we investigate the rate of BRCA2 mutation in sporadic breast cancers and in a set of cell lines that represent twelve other tumour types. Surprisingly, mutations in BRCA2 are infrequent in cancers including breast carcinoma. However, a probable germline mutation in a pancreatic tumour cell line suggests a role for BRCA2 in susceptibility to pancreatic cancer.

BRCA2 Protein↗
PubMed1995

Genomic structure, expression and mutational analysis of the P15 (MTS2) gene.

The P15 gene (MTS2) encodes a cyclin-dependent kinase (CDK) inhibitor with considerable sequence identity and biochemical similarity to the CDK inhibitor p16. It is closely linked to the P16 gene (MTS1) and is homozygously deleted in many tumor cell lines. These features suggest that p15 may be a tumor suppressor. We have determined the genomic structure of P15 and examined its pattern of mRNA expression. In addition, we have shown that ectopic expression of p15 inhibits growth of tumor-derived cell lines. We have also searched for P15 mutations in tumor cell lines and in 9p21-linked melanoma kindreds. Other than the previously described homozygous deletions, no mutations of P15 were found. Collectively, these observations suggest a role for p15 in growth regulation, but a limited role for p15 in tumor progression.

Base Sequence↗
PubMed1995

Complex structure and regulation of the P16 (MTS1) locus.

The p16 gene (P16, MTS1, CDKN2) encodes a negative regulator of the cell cycle. Molecular genetic techniques have been used to explore the role of p16 in normal development and cancer. Two transcripts derived from the p16 gene with distinct protein coding potentials are described. The previously undescribed transcript form has the same exons 2 and 3 as the p16-encoding mRNA but contains a different exon 1. The human p16 transcripts are detected in various tissues, and the ratio of the transcripts is regulated in both a tissue-specific and cell cycle-specific manner. The P16-derived mRNAs are probably generated from separate promoters, and transcription from one of the promoters appears to be regulated, at least in part, by the retinoblastoma gene product.

Base Sequence↗
PubMed1995

Differential inactivation of CDKN2 and Rb protein in non-small-cell and small-cell lung cancer cell lines.

BACKGROUND: The CDKN2 gene encodes the human cyclin-dependent kinase 4 inhibitor. This inhibitor protein is believed to be a tumor suppressor that plays an essential role in cell cycle regulation. One half of all cancer cell lines and one fourth of lung cancer cell lines examined to date contain homozygous deletions (i.e., both alleles lost) of CDKN2. However, the relative frequency of homozygous CDKN2 deletions in non-small-cell lung cancers (NSCLC) and in small-cell lung cancers (SCLC) has not been determined. Inactivation or loss of another tumor suppressor encoded by the retinoblastoma gene (the Rb protein) is more common in SCLC than in NSCLC. PURPOSE: We measured the frequency of homozygous CDKN2 deletions in 77 NSCLC and in 93 SCLC tumor cell lines. In addition, possible associations were explored between CDKN2 gene loss, the presence or absence of Rb protein, and the clinical status of lung cancer patients. METHODS: DNA was isolated from each tumor cell line and from the primary tumor and normal tissue of one NSCLC patient. Sequences corresponding to exons 1 and 2 of the CDKN2 gene were amplified by use of the polymerase chain reaction, and the resulting amplification products were analyzed by agarose gel electrophoresis and DNA blotting. Genomic DNA blotting was also used to evaluate CDKN2 gene deletions. The frequency of homozygous CDKN2 loss and the presence or absence of functional Rb protein (reported previously) in the cell lines were compared. RESULTS: Homozygous deletion of CDKN2 was detected in 18 (23%) of 77 cell lines established from patients with NSCLC, compared with one (1%) of 93 cell lines established from patients with SCLC (P < .001). No CDKN2 gene loss was observed in the normal tissue of an NSCLC patient whose tumor cell line showed homozygous deletion of the gene; however, the primary tumor from this patient had evidence of CDKN2 loss. Homozygous CDKN2 deletion was detected in 13 (28%) of 46 tumor cell lines from patients with stage III or stage IV NSCLC, compared with zero of 10 tumor cell lines from patients with stage I or stage II NSCLC. Coincident loss of CDKN2 genes and functional Rb protein was rarely observed (in two of 135 cell lines). CONCLUSION: The frequency of homozygous CDKN2 gene deletion in NSCLC cell lines is greater than that observed for any other known, or candidate, tumor suppressor gene. IMPLICATION: Further study of the role of CDKN2 gene alteration in the pathogenesis of NSCLC is needed.

Base Sequence↗
PubMed1995

Software trapping: a strategy for finding genes in large genomic regions.

We present an approach to the gene identification phase of positional cloning that combines sparse sampling of DNA sequences from large genomic regions with computational analysis. We call the method "software trapping." The goal is to find coding exons while avoiding massive DNA sequence determination and contig assembly. Instead, rapid sequence sampling is combined with exon screening software such as a newly developed package called XPOUND to identify coding sequences. We have tested the approach using a set of model genomic sequences with known intron/exon structures as well as with bona fide P1 genomic clones. The results suggest that the strategy is a useful complement to other methods for finding genes in poorly characterized regions of genomes.

Bacteriophage P1↗
PubMed1995

Comparative analysis of Homo sapiens and Mus musculus cyclin-dependent kinase (CDK) inhibitor genes p16 (MTS1) and p15 (MTS2).

Cyclin-dependent kinase inhibitors are a growing family of molecules that regulate important transitions in the cell cycle. At least one of these molecules, p16, has been implicated in human tumorigenesis while its close homolog, p15, is induced by cell contact and transforming growth factor-beta (TGF-beta). To investigate the evolutionary and functional features of p15 and p16, we have isolated mouse (Mus musculus) homologs of each gene. Comparative analysis of these sequences provides evidence that the genes have similar functions in mouse and human. In addition, the comparison suggests that a gene conversion event is part of the evolution of the human p15 and p16 genes.

Amino Acid Sequence↗
PubMed1995

Monitoring the efficacy of hybrid selection during positional cloning: the search for BRCA1.

Positional cloning often requires isolation of candidate genes from a large, genetically defined region. Hybrid selection (direct cDNA selection, solution hybrid capture) is a rapid, simple procedure that has been used to identify expressed sequence tags (ESTs) from cloned genomic DNA. We used hybrid selection to screen a 600-kb region that includes the BRCA1 gene. From a set of 931 sequenced clones, we obtained 118 nonoverlapping candidate ESTs from ovary and lymphocyte cDNA. We analyzed the results of our hybrid selection experiments with particular attention to the overall completeness, efficiency, and background noise of the experiment. We introduce simple parameters that serve as measures of important aspects of the hybrid selection process in the context of positional cloning.

BRCA1 Protein↗
PubMed1995

Cell-cycle regulators and cancer.

Cancer is a disease characterized by loss of cellular growth control. As such, it is not surprising that the molecular machinery of the cell cycle is involved in tumorigenesis. Recent discoveries have brought several cell-cycle regulators into sharp focus as factors in human cancer. Among the most conspicuous types of molecule to emerge from ongoing studies in this field are the cyclin-dependent kinase inhibitors such as p16. These molecules have several hallmarks of tumor suppressors and are perfectly positioned to regulate critical decisions in cell growth. The P16 gene appears to be a particularly significant target for mutation in sporadic tumors and in at least one form of hereditary cancer.

Carrier Proteins↗
PubMed1995

Homozygotes for CDKN2 (p16) germline mutation in Dutch familial melanoma kindreds.

The p16 gene (CDKN2) which is localized on chromosome 9p21, is deleted in a significant number of sporadic cancers. Moreover, germline mutations identified in some melanoma-prone kindreds last year suggested that CDKN2 is identical to the 9p21-linked melanoma susceptibility gene (MLM); however, failure to identify p16 mutations in all melanoma kindreds putatively linked to 9p21 left some doubts. We have analysed CDKN2 coding sequences in 15 Dutch familial atypical multiple mole-melanoma (FAMMM) syndrome pedigrees, and identified a 19 basepair (bp) germline deletion in 13 of them. All 13 families originate from an endogamous population. The deletion causes a reading frame shift, predicted to result in a severely truncated p16 protein. Interestingly, two family members are homozygous for the deletion, one of whom shows no obvious signs of disease. This surprising finding demonstrates that homozygotes for this CDKN2 mutation are viable, and suggests the presence of a genetic mechanism that can compensate for the functional loss of p16. Our results also greatly strengthen the notion that p16 is indeed MLM.

Amino Acid Sequence↗
PubMed1995

Efficient, automatic detection of heterozygous bases during large-scale DNA sequence screening.

A crucial factor in the success of positional cloning efforts is the ability to screen rapidly many different candidate genes for mutations. By modifying standard software, we have improved the detection of heterozygous base positions in PCR products sequenced by cycle sequencing. A key element of the method is the incorporation of a modified heterozygote detection algorithm that permits the use of DNA sequence data derived from PCR and sequencing reactions that have not been fully optimized. This allows sequencing runs of average quality to be used. We demonstrate that the sensitivity and specificity of the method are well suited to mutation detection applications such as positional cloning.

Algorithms↗
PubMed1994

Localization of the VHR phosphatase gene and its analysis as a candidate for BRCA1.

The VH1-related human protein (VHR) gene was localized to human chromosome 17q21 in a region thought to contain the BRCA1 locus, a locus that confers susceptibility to breast and ovarian cancer. VHR encodes a phosphatase with dual specificity for tyrosine and serine residues. Thus it is a plausible candidate for a tumor suppressor gene such as BRCA1. To test this possibility, the VHR coding sequence was screened in individuals with familial breast cancer and in sporadic breast tumor and breast cancer cell lines. No mutations were detected, suggesting that the VHR gene is not BRCA1.

BRCA1 Protein↗
PubMed1994

Localization of the 9p melanoma susceptibility locus (MLM) to a 2-cM region between D9S736 and D9S171.

A familial melanoma susceptibility locus (MLM) was identified on the short arm of chromosome 9 in a set of Utah and Texas kindreds. Subsequent confirmation was reported for a set of 26 Australian kindreds, a set of 7 Dutch kindreds, and a set of 13 NIH melanoma kindreds. The original localization report placed the locus near the IFNA-D9S126 interval on chromosome 9p. We report further localization using D9S171 and a newly developed marker, D9S736, both of which lie in the IFNA-D9S126 interval. Analysis of this set of kindreds places the melanoma susceptibility locus in a 2-cM region that is proximal to D9S736 and distal to D9S171.

Adult↗
PubMed1994

Localization of a putative tumor suppressor gene by using homozygous deletions in melanomas.

The p21 region of human chromosome 9 is thought to contain a gene (MLM) involved in genetic susceptibility to melanoma and a gene or genes that influence progression of certain other tumors. Genomic clones that span a large region in 9p21 surrounding the presumptive tumor suppressor gene(s) have been isolated. A set of sequence-tagged sites in this region has been developed. By using these markers and others previously reported, the 9p21 region has been studied by physical mapping in 84 melanoma cell lines. A putative tumor suppressor gene, perhaps MLM itself, has been localized to a region of less than 40 kb that lies proximal (centromeric) to the alpha-interferon gene cluster.

Chromosome Mapping↗
PubMed1994

A cell cycle regulator potentially involved in genesis of many tumor types.

A putative tumor suppressor locus on the short arm of human chromosome 9 has been localized to a region of less than 40 kilobases by means of homozygous deletions in melanoma cell lines. This region contained a gene, Multiple Tumor Suppressor 1 (MTS1), that encodes a previously identified inhibitor (p16) of cyclin-dependent kinase 4. MTS1 was homozygously deleted at high frequency in cell lines derived from tumors of lung, breast, brain, bone, skin, bladder, kidney, ovary, and lymphocyte. Melanoma cell lines that carried at least one copy of MTS1 frequently carried nonsense, missense, or frameshift mutations in the gene. These findings suggest that MTS1 mutations are involved in tumor formation in a wide range of tissues.

Base Sequence↗