Search PubMed⌕ Search

Biomedical subjects

R Callahan

Publications and source records attributed to R Callahan.

At least 37 records · Page 2Linked to original sources
PubMed1995

The chromosomal location of the mouse mammary tumor gene Int6 and related pseudogenes in the mouse genome.

The Int6 gene is a common insertion site for the mouse mammary tumor virus (MMTV) in mouse mammary tumors. We have determined that this gene is located centromeric of the Myc protooncogene on mouse chromosome 15. In the mouse genome there are several other Int6-reactive restriction fragments that are located on mouse chromosomes 6, 11, 14, 17, and 18. Nucleotide sequence analysis of four of six of these additional Int6 fragments showed that they contain processed Int6 pseudogenes. Comparisons between the Int6 genes of the inbred mouse laboratory strains and the wild mouse species Mus spretus and Mus mus musculus indicate that some pseudogenes were present before divergence of these species and others were acquired since their separation.

Animals↗
PubMed1995

A novel non-mouse mammary tumor virus activation of the Int-3 gene in a spontaneous mouse mammary tumor.

In a mouse mammary tumor model system in which carcinogenic progression can be investigated, we have found a unique mutation of Int-3 associated with progression from premalignant lobular hyperplasia to tumor. Sequence analysis of the rearranged fragment revealed an insertion of an intracisternal type A particle (IAP) within the Int-3 gene. Int-3 is mutated frequently in mouse mammary tumor virus (MMTV)-induced mammary tumors by insertion of MMTV proviral DNA into this intragenic region. In these mutations, the insertion produces a chimeric Int-3 transcript encoding the cytoplasmic portion of the Int-3 protein driven by the MMTV long terminal repeat promoter. In this case, the IAP DNA was inserted in the opposite transcriptional orientation relative to Int-3; nevertheless, a similar chimeric RNA transcript driven by a cryptic promoter in the oppositely oriented 5' IAP long terminal repeat was generated. This is the first demonstration that an insertional mutation unrelated to MMTV activates an Int gene commonly associated with mammary tumorigenesis.

Animals↗
PubMed1995

Int-6, a highly conserved, widely expressed gene, is mutated by mouse mammary tumor virus in mammary preneoplasia.

With a unique mouse mammary tumor model system in which mouse mammary tumor virus (MMTV) insertional mutations can be detected during progression from preneoplasia to frank malignancy, including metastasis, we have discovered a new common integration site (designated Int-6) for MMTV in mouse mammary tumors. MMTV was integrated into Int-6 in a mammary hyperplastic outgrowth line, its tumors and metastases, and two independent mammary tumors arising in unrelated mice. The Int-6 gene is ubiquitously expressed as a 1.4-kb RNA species in adult tissues and is detected beginning at day 8 of embryonic development. The nucleotide sequence of Int-6 is unrelated to any of the known genes in the GenBank database. MMTV integrates within introns of the gene in the opposite transcriptional orientation. In each tumor tested, this results in the expression of a truncated Int-6/long terminal repeat (LTR) chimeric RNA species which is terminated at a cryptic termination signal in the MMTV LTR. Since the nonrearranged Int-6 alleles in these tumors contain no mutations, we favor the conclusion that truncation of the Int-6 gene product either biologically activates its function or represents a dominant-negative mutation.

Amino Acid Sequence↗
PubMed1995

Constitutive expression of a truncated INT3 gene in mouse mammary epithelium impairs differentiation and functional development.

INT3 is interrupted by retroviral DNA insertion in approximately 18% of primary Czech mouse mammary tumors induced by mouse mammary tumor virus. One consequence of these insertions is the production of a 2.4-kilobase, tumor-specific RNA transcript encoding the entire intracellular domain of the Int3 protein which is initiated from the 3' long terminal repeat promoter of the inserted viral genome. Female mice (FVB-3) transgenic for a genomic fragment comprised of this truncated region of INT3 express the 2.4-kilobase truncated INT3 transcript and exhibit focal mammary tumors at 100% penetrance. INT3 is a member of a family of genes, highly conserved through evolution and characterized by Drosophila melanogaster Notch and Caenorhabditis elegans lin-12, the function of which relates to cell fate determination. Upon transfection into the appropriate hosts, expression vectors of truncated Notch and lin-12, representing their respective cytoplasmic domains, have been demonstrated to effect their complete gene function with respect to cell fate determination. This suggests that the extracellular portion of these proteins function only to regulate activity. Reciprocal transplantation of transgenic FVB-3 and normal mammary tissue to the epithelium-divested fat pads of the respective donor females demonstrates that FVB-3 mammary epithelium is unable to grow and/or to functionally differentiate. However, normal epithelium grows and fully differentiates in transgenic FVB-3 fat pads, indicating that the dysfunction of FVB-3 mammary glands is due to a deficiency inherent in their epithelium. Electron microscopy reveals that transgenic INT3 epithelial cells do not form intercellular junctional complexes in the developing subadult mammary gland. The hormonal stimulation of pregnancy overcomes the deficiency for ductal growth so apparent in the virgin gland such that pregnant FVB-3 glands produce complete ductal systems. Nevertheless, during pregnancy, FVB-3 mammary cells fail to form secretory lobules and to produce milk. Examination of INT3 expression by immunocytochemistry and reverse transcriptase-PCR show that INT3 is expressed constitutively in mammary stroma and epithelia at all stages of postpubertal mammary evolution. These results indicate that deregulated expression of a truncated Int3 in mammary epithelial cells limits their capacity to perform the cell fate decisions required for morphogenesis and functional differentiation.

Amino Acid Sequence↗
PubMed1995

Detection of DNA mutations in acid formalin-fixed paraffin-embedded archival tumor specimens by polymerase chain reaction-single strand conformation polymorphism analysis.

A single strand conformation polymorphism analysis on polymerase chain reaction (PCR)-amplified genomic DNA was used for the detection of DNA mutations in acid formalin-fixed, paraffin-embedded tissues. Fifty non-small cell lung cancers were screened for mutations in the exon 5 of the p53 tumor suppressor gene. Structural abnormalities of the gene were found in nine (18%) of the tumors examined. The results show that paraffin-embedded tissues fixed in unbuffered formalin can be a good source of DNA for a mobility shift analysis by neutral polyacrylamide gel electrophoresis. The described procedure allows retrospective genetic studies on paraffin blocks available in all pathology departments.

Base Sequence↗
PubMed1994

Evidence for involvement of BRCA1 in sporadic breast carcinomas.

The hereditary breast cancer gene BRCA1 previously has been localized to chromosome 17q21. We looked for evidence of involvement of this region of chromosome 17 in 130 sporadic breast cancers. Seventeen polymorphic sequence tagged site markers were examined in these tumors between the D17S250 and D17S579 loci to screen for deletions as measured by loss of heterozygosity. The smallest common region that was deleted occurred in the approximately 120-kilobase interval between the D17S846 and D17S746 loci within the BRCA1 region. Delineation of this commonly deleted area should accelerate attempts to identify the involved gene(s) and its relationship to BRCA1.

Breast Neoplasms↗
PubMed1994

Evidence for a second tumor suppressor gene on 17p linked to high S-phase index in primary human breast carcinomas.

The short area of chromosome 17 is a frequent target for deletions in human tumors, including breast cancer. We have investigated by restriction fragment polymorphism analysis the pattern of loss of heterozygosity (LOH) at four loci on 17p13.1-17pter in a panel of 110 primary human breast carcinomas. A copy of the p53 gene was lost in 23% of the informative cases. Point mutations in the p53 gene were statistically associated with LOH at the same locus (p = 0.003) but not at other loci on 17p13.3-17pter. A second region bordered by the loci D17S5/D17S28 (17p13.3) and D17S34 (17pter) is also affected by LOH, independent of point mutations in the p53 gene. We propose the presence of a second tumor suppressor gene within this region. In support of this hypothesis is the significant association (p = 0.005) between LOH at the D17S5/D17S28, but not at the TP53 or D17S34 loci, and tumors having a high S-phase index.

Alleles↗
PubMed1994

[Demonstration of two regions involved in chromosome 1p deletion in breast tumors].

Alteration of chromosome 1 is the most consistent cytogenetic abnormality found in human breast carcinoma. Cytogenetic studies have shown independent alterations on the two arms of chromosome 1; increased copy number of the long arm and loss of the short arm of chromosome 1. We carried out deletion analysis of the 1p region by using restriction fragment length polymorphism markers mapping to the long (six markers) and short arm (22 markers). Thirty-five of the 74 (47%) human breast tumors tested showed somatic loss of heterozygosity at one or more loci on the short arm. Two commonly deleted regions, 1p13-p21 and 1p32-pter, were identified. Our findings suggest that two tumor suppressor genes involved in the development of human breast carcinoma may occur on the short arm of the chromosome 1.

Alleles↗
PubMed1994

Growth suppression of normal mammary epithelial cells by wild-type p53.

p53 mutations are frequent in human breast cancer. In order to understand the role of p53 in the context of the accumulation of mutations in breast cancer, a model of non transformed mammary cells was sought. The HC11 cells are immortalized, non transformed rodent mammary epithelial cells which synthesize milk proteins following stimulation with lactogenic hormones. p53 protein was readily detected in HC11 protein extracts with the PAb421 antibody. Two mutations were identified in the p53 cDNA from HC11 cells: a missense mutation at codon 138, substituting Trp for Cys, and a microdeletion, codon 123 to 130, of exon 5. The latter results from an intronic mutation of the splice acceptor site at the intron 4/exon 5 junction. The mutations affect separate p53 alleles, and no wt allele was found. Wt p53 was introduced into HC11 cells by means of a retroviral vector, under the control of a Cd(++)-inducible promoter. In the presence of CdSO4 a dramatic growth inhibition was observed. A temperature-sensitive mutant p53 gene was also transfected into HC11 cells. This resulted in a marked inhibition of cells growth at 32 degrees C, when the p53 is in the wt conformation, while no effect was observed at 37 degrees C, when the mutant conformation is predominant. wt p53-mediated inhibition of monolayer growth does not involve induction of programmed cell death and does not activate de novo synthesis of differentiation-specific milk proteins. We conclude that mutations in the p53 gene likely played a role in their immortalization. The HC11 cells provide a model for assessing the cooperative action of other mutations in mammary tumorigenesis.

Alleles↗
PubMed1993

Identification of three regions on chromosome 17q in primary human breast carcinomas which are frequently deleted.

We have examined the long arm of chromosome 17 in sporadic breast carcinomas for the loss of heterozygosity (LOH) at 18 polymorphic loci. At least three distinct regions could be identified by the frequency of LOH and confirmed by high density deletion maps of individual tumor DNAs. A proximal region affected by LOH is located in a 22-cM region defined by D17S73 and NME1 and thus is similar in location to the region thought to contain the BRCA1 gene associated with familial breast and breast/ovarian cancer. The central region affected by LOH is bordered by the D17S86 and D17S21 loci and is estimated to be 28 cM in size. The third region is bordered by the D17S20 and D17S77 loci which are 11 cM apart. These results define three independent regions of chromosome 17q which are likely to contain tumor suppressor genes relevant to the etiology of sporadic breast carcinoma.

Breast Neoplasms↗
PubMed1993

Growth suppression of normal mammary epithelial cells by wild-type p53.

We examined the status of the p53 gene in the HC11 normal mammary epithelial cells. Two mutations were identified: a Cys to Trp change at codon 138 and a microdeletion of codon 123 to 130 resulting from mutation of the splice acceptor site. These two mutations were independent, and no wild-type p53 allele was found. Introduction of wt-p53 strongly inhibited growth in monolayer. Thus, the absence of wt-p53 can be sufficient for the immortalization of mammary cells.

Amino Acid Sequence↗
PubMed1993

p53 mutations and histological type of invasive breast carcinoma.

A polymerase chain reaction-single strand conformation polymorphism assay was used to assess p53 mutations in 148 invasive breast carcinomas, selected on the basis of their histotype. They comprised 56 lobular, 47 ductal, 19 mucinous, 18 medullary, and 8 papillary carcinomas. The distribution of p53 mutations was significantly different (P = 0.006) in the histotypes examined: mutations were frequent in medullary (39%) and ductal (26%), less common in lobular (12%), and absent in mucinous and papillary carcinomas. The frequency of mutations in the exon 5 of the p53 gene was significantly higher in medullary carcinomas than in the other histotypes: 5 (63%) of the mutations found in exon 5 were observed in medullary carcinomas (P = 0.012). One hundred twenty-two tumors from the total were also examined by immunohistochemistry for p53 overexpression using antibody PAb 1801. A specific immunostaining in neoplastic cells was present in 12 tumors. A strong correlation (P < 0.001) was observed between p53 mutations and nuclear accumulation of the p53 protein: 10 tumors were scored positive for both p53 mutation and overexpression. However, in 9 cases having a mutated p53 gene we failed to find a positive immunoreaction. A significant association (P = 0.01) was present between mutations in the p53 gene and high proliferative activity of the tumors determined by immunohistochemistry with monoclonal antibody Ki-67. Moreover, a significantly higher expression of the Ki-67 antigen was found in medullary carcinomas compared to the other histotypes. Our findings indicate that in invasive breast carcinomas structural abnormalities of the p53 gene are mainly seen in medullary and ductal tumors and that the other histological types, especially those associated with a high level of differentiation and favorable prognosis, show a very low incidence of p53 mutations.

Base Sequence↗
PubMed1993

Genetic and molecular heterogeneity of breast cancer cells.

We have undertaken a systematic study of primary human breast tumor DNAs to identify and characterize frequently occurring somatic mutations. Loss of heterozygosity (LOH) was found on chromosomes 1p (37%), 1q (20%), 3p (30%), 7 (41%), 13q (30%), 17p (49%), 17q (29%) and 18q (34%) in our tumor DNA panel. Specific subsets of tumors could be defined based on the particular collection of mutations they contained. One goal of these studies has been to determine whether there is a significant association between specific mutations and clinical parameters of the disease. We have found that LOH on chromosome 17p in tumor DNAs is associated with breast tumors having a high proliferative index and that LOH on chromosome 7 is associated with patients having a poor prognosis. Our analysis of chromosome 17 suggests that there may be as many as four tumor suppressor genes affected in primary human breast tumors.

Animals↗
PubMed1993

In primary human breast carcinomas mutations in exons 5 and 6 of the p53 gene are associated with a high S-phase index.

A series of 121 human breast tumors was screened for point mutations in exons 5 through 8 of the p53 gene, by SSCP analysis. On the same tumor samples, the S-phase index (SPI) was determined by the incorporation of BUdR in fresh tissue. p53 mutations were observed in 29% of the cases. The frequency of point mutations for the individual exons was: exon 5, 10.0%; exon 6, 9.9%; exon 7, 7.1% and exon 8, 5.5%. Two mutations detected by SSCP were confirmed by sequencing the p53 cDNA. The presence of a p53 mutation, irrespective of its location, correlates (p = 0.003) with a high SPI. This association appears to primarily reflect mutations in exon 5 (p = 0.0002) and exon 6 (p = 0.05), since mutations in exons 7 and 8 failed to show any association. These results indicate that mutations in the p53 gene identify highly proliferating tumors, and that the position of the p53 mutation may have different effects upon the proliferative activity of tumor cells in vivo.

Base Sequence↗