Biomedical subjects

R Callahan

Publications and source records attributed to R Callahan.

At least 19 recordsLinked to original sources

PubMed2006

Kit and PDGFR-alpha activities are necessary for Notch4/Int3-induced tumorigenesis.

Transgenic mice overexpressing Notch4 intracellular domain (Int3) under the control of the whey acidic protein (WAP) or mouse mammary tumor virus-long terminal repeat promoters, develop mammary tumors. Microarray analysis of these tumors revealed high levels of c-Kit expression. Gleevec is a tyrosine kinase inhibitor that targets c-Kit, platelet-derived growth factor receptors (PDGFRs) and c-Abl. This led us to speculate that tyrosine kinase receptor activity might be a driving force in the development of Int3 mammary tumors. WAP-Int3 tumor-bearing mice were treated with continuous release of Gleevec using subcutaneously implanted Alzet pumps. Phosphorylation of c-Kit, PDGFRs and c-Abl is inhibited in Int3 transgenic mammary tumors by Gleevec. Inhibition of these enzymes is associated with a decrease in cell proliferation and angiogenesis, and an induction of apoptosis. To examine the signaling mechanisms underlying Notch4/Int3 tumorigenesis, we employed small interfering RNA (siRNA) to knock down c-Kit, PDGFRs and c-Abl alone or in combination and observed the effects on soft agar growth of HC11 cells overexpressing Int3. Only siRNA constructs for c-Kit and/or PDGFR-alpha were able to inhibit HC11-Int3 colony formation in soft agar. Our data demonstrate an inhibitory effect of Gleevec on Int3-induced transformation of HC11 cells and mammary tumors and indicate an oncogenic role for c-Kit and PDGFR-alpha tyrosine kinases in the context of Int3 signaling.

Animals↗

PubMed2004

Candidate target genes for loss of heterozygosity on human chromosome 17q21.

Loss of heterozygosity (LOH) on chromosome 17q21 has been detected in 30% of primary human breast tumours. The smallest common region deleted occurred in an interval between the D17S746 and D17S846 polymorphic sequences tagged sites that are located on two recombinant P1-bacteriophage clones of chromosome 17q21: 122F4 and 50H1, respectively. To identify the target gene for LOH, we defined a map of this chromosomal region. We found the following genes: JUP, FK506BP10, SC65, Gastrin (GAS) and HAP1. Of the genes that have been identified in this study, only JUP is located between D17S746 and D17S846. This was of interest since earlier studies have shown that JUP expression is altered in breast, lung and thyroid tumours as well as cell lines having LOH in chromosome 17q21. However, no mutations were detected in JUP using single-strand conformation polymorphism analysis of primary breast tumour DNAs having LOH at 17q21. We could find no evidence that the transcription promoter for JUP is methylated in tumour DNAs having LOH at 17q21. We suspect that the target gene for LOH in primary human breast tumours on chromosome 17q21 is either JUP and results in a haploinsufficiency for expression or may be an unidentified gene located in the interval between D17S846 and JUP.

Breast Neoplasms↗

PubMed2001

Evidence for the transforming activity of a truncated Int6 gene, in vitro.

Int6/eIF3-p48 was first identified as a common integration site for MMTV in mouse mammary tumors. In all cases, the MMTV integration event resulted in an interruption of the normal Int6 transcript from one allele leaving the second allele intact and operative. We hypothesize that insertion of MMTV into Int6 results in a mutated allele that encodes a shortened Int6 mRNA and protein (Int6sh), which either modifies normal Int6 function or possesses a new independent function. To confirm the transforming potential of the mutation and its dominant function, we transfected two mammary epithelial cell lines, MCF10A (human), and HC11 (mouse), with Int6sh under the control of the elongation factor-1alpha (eEF1A) promoter. Expression of Int6sh in MCF10A and HC11 mammary epithelial cells leads to anchorage-independent growth in soft agar indicative of a transformed phenotype. Colonies selected from agar exhibited high levels of mutated Int6sh and wild type Int6 RNA transcripts by RT-PCR and Northern blot analysis. In addition, Int6sh transformed MCF10A and HC11 cells formed nodular growths, in vivo, in immune compromised hosts. NIH3T3 cells, mouse embryo fibroblasts, were also transformed to anchorage-independent growth in vitro by Int6sh expression. These observations provide direct evidence that the Int6 mutations observed in MMTV-induced tumors and hyperplasia contribute to the malignant transformation of the mammary epithelial cells.

3T3 Cells↗

PubMed2001

Notch signaling in mammary gland tumorigenesis.

The Notch receptor protein and its signaling pathway have been well conserved throughout evolution and appear to be pivotal components in cell fate decisions during development. Recent studies suggest that, depending on the cellular and developmental context, Notch signaling may also affect cell proliferation and programmed cell death. Mammals have four related Notch genes. One of these, designated Notch-4, was found to be a common integration site for the mouse mammary tumor virus in mouse mammary tumors. One consequence of this type of viral integration event is the ectopic expression of the intracellular domain of Notch-4 that corresponds to a gain-of-function mutation. Expression of "activated" Notch-4 in mammary epithelium has profound effects on mammary gland development and tumorigenesis. In this review, we briefly summarize the structure and function of the Notch receptor, as well as the components that comprise and modify the signaling pathway. Finally we discuss the potential role of Notch in mammary gland development and tumorigenesis.

Animals↗

PubMed2001

Reduced expression of INT-6/eIF3-p48 in human tumors.

The int-6 gene, originally identified as a common integration site for the mouse mammary tumor virus (MMTV) in mouse mammary tumors, encodes the p48 component of the eukaryotic translation initiation factor-3 (eIF3-p48). Int-6/eIF3-p48 is expressed in all adult tissues which have been tested and early in embryonic development. Int-6/eIF3-p48 has been highly conserved throughout evolution and the deduced amino acid sequence of the human gene product is identical to the mouse protein. Viral insertions at the Int-6/eIF3-p48 locus in mouse mammary tumors result in production of chimeric Int-6/eIF3-p48/MMTV products that may act as dominant negative oncoproteins. Int-6/eIF3-p48 has also been identified as a human protein that binds to the human T-cell leukemia virus type I Tax oncoprotein. The role of Int-6/eIF3-p48 in human carcinogenesis is unknown at the present time. In this study we have examined Int-6/eIF3-p48 gene status and expression in two of the most common forms of cancer in humans, breast and lung tumors. Sixty-two breast carcinomas and 78 non-small cell lung carcinomas (NSCLC) were investigated. LOH at the Int-6/eIF3-p48 locus was observed in 5 (21%) of 24 informative breast tumors and 10 (29%) of 34 informative lung tumors. A reduced expression of Int-6/eIF3-p48 was seen in 23 (37%) of breast cancer samples and 24 (31%) of NSCLC samples. An association between Int-6/eIF3-p48 expression and LOH at the Int-6/eIF3-p48 locus was observed. Int-6/eIF3-p48 expression was not related to commonly used pathological parameters in breast cancer patients, while in NSCLC patients int-6/eIF3-p48 expression was mainly seen in adenocarcinomas (P<0.0001). In conclusion, our data show for the first time a decreased expression of Int-6/eIF3-p48 in a consistent portion of human breast and lung carcinomas, frequently associated with LOH at the Int-6/eIF3-p48 locus. Additional studies on larger series of tumor specimens with long-term follow-up are needed to determine whether Int-6/eIF3-p48 expression may represent a new prognostic or predictive marker.

Biomarkers, Tumor↗

PubMed2000

Notch signaling is essential for vascular morphogenesis in mice.

The Notch gene family encodes large transmembrane receptors that are components of an evolutionarily conserved intercellular signaling mechanism. To assess the role of the Notch4 gene, we generated Notch4-deficient mice by gene targeting. Embryos homozygous for this mutation developed normally, and homozygous mutant adults were viable and fertile. However, the Notch4 mutation displayed genetic interactions with a targeted mutation of the related Notch1 gene. Embryos homozygous for mutations of both the Notch4 and Notch1 genes often displayed a more severe phenotype than Notch1 homozygous mutant embryos. Both Notch1 mutant and Notch1/Notch4 double mutant embryos displayed severe defects in angiogenic vascular remodeling. Analysis of the expression patterns of genes encoding ligands for Notch family receptors indicated that only the Dll4 gene is expressed in a pattern consistent with that expected for a gene encoding a ligand for the Notch1 and Notch4 receptors in the early embryonic vasculature. These results reveal an essential role for the Notch signaling pathway in regulating embryonic vascular morphogenesis and remodeling, and indicate that whereas the Notch4 gene is not essential during embryonic development, the Notch4 and Notch1 genes have partially overlapping roles during embryogenesis in mice.

Age Factors↗

PubMed2000

MMTV-induced mammary tumorigenesis: gene discovery, progression to malignancy and cellular pathways.

The study of the mouse mammary tumor virus (MMTV) has provided important insights into the mechanisms of gene transcription regulation by steroid hormones, the mode of action of heritable super antigens and the progressive nature of neoplastic transformation in the mammary gland. Here we describe the current situation with respect to the latter aspect of MMTV biology and the prospects for further advance in our understanding of breast cancer in humans that may be expected from a continued study of MMTV-induced mammary neoplasia. MMTV is a heritable somatic mutagen whose target range is limited. Commonly, the tumorigenic capacity of MMTV is restricted to mammary gland, whereas infection is found in a variety of cell types. In order to replicate, proviral DNA must be inserted into the cell DNA and cell division is required to fix the mutation. Yet only in the mammary epithelium does this lead to neoplastic transformation. This suggests a unique relationship between MMTV and mammary epithelium. In evaluating this relationship, we and others have discovered genes and potential gene pathways that are pertinent in mammary differentiation and neoplasia. In addition, the clonal nature of these progressive events from normal to malignant phenotype has become increasingly clear. The weight of these observations compel us to conclude that mammary neoplasms arise from multipotent mammary epithelial cells through a process of acquired mutations that are reflected in the increasingly malignant nature of the population of progeny produced by these damaged stem cells.

Animals↗

PubMed2000

Identification of a novel NOTCH-4/INT-3 RNA species encoding an activated gene product in certain human tumor cell lines.

Ectopic expression of the intracellular domain of NOTCH-4/INT-3 leads to tumorigenesis in the mouse mammary gland. This results from a gain-of-function mutation. To evaluate gain-of-function NOTCH-4/INT-3 activity in human cancers we have surveyed human breast, lung, and colon carcinoma tissue culture cell lines for evidence of increased NOTCH-4/INT-3 RNA expression. High levels of a 1.8 Kb NOTCH-4/INT-3 RNA species are detected in normal human testis but not in other tissues where a 6.5 Kb species is prevalent. Transformed human cancer cell lines express the 1.8 Kb NOTCH-4/INT-3 RNA species. We show that this RNA species encodes a truncated form of the NOTCH-4/INT-3 intracellular domain (ICD). This novel NOTCH-4/INT-3 protein includes the CDC10 repeats and amino acid residues C-terminal to them, but is missing the CBF-1 binding region of the NOTCH-4/INT-3 ICD. This suggests that it has a different mode of action. Furthermore, we show that a transgene which expresses the 1.8 Kb NOTCH-4/INT-3 RNA species in the 'normal' human mammary epithelial cell line MCF-10A enables these cells to grow in soft agar.

Amino Acid Sequence↗

PubMed1999

Characterization of the Drosophila ortholog of mouse eIF-3p48/INT-6.

The mouse mammary tumor virus (MMTV) has been shown to integrate frequently into INT-6 in MMTV-induced mouse mammary tumors. The INT6 gene has been highly conserved through evolution and has recently been shown to encode the p48 component of the eucaryotic translation initiation factor 3 (eIF-3) complex. We report here the isolation of the Drosophila eIF-3p48/INT-6. The gene comprises three exons within 1.8kb of genomic DNA located at cytogenetic position 73C2 in the Drosophila genome. The 1.5kb eIF-3p48/INT-6 RNA species encodes a protein composed of 364 amino-acid residues whose sequence is 71% similar to that of the mouse/human eIF-3/INT-6 amino-acid sequence. eIF-3p48/INT-6 RNA is expressed throughout development in Drosophila and the encoded protein is associated with the microsomal subcellular fraction.

Amino Acid Sequence↗

PubMed1999

Prognostic significance of loss of heterozygosity at loci on chromosome 17p13.3-ter in sporadic breast cancer is evidence for a putative tumour suppressor gene.

Several studies indicate that the short arm of chromosome 17 is one of the most frequently altered regions in sporadic breast carcinomas (45-60%). In the present report the 17p13.3-ter locus in tumour DNA of breast cancer patients, along with their matching normal lymphocyte DNA, have been mapped with four markers (D17S5, D17S379, ABR and D17S34), spanning nearly 3 cM of the telomer. Sixty-five of 143 heterozygous tumours had lost at least one of the markers at the minimum region of loss (45%). High levels of loss of these distal markers on 17p13.3 are independent of TP53 mutations and are associated with tumour cell proliferation. A follow-up period of over 7 years demonstrates that loss of these markers correlates both with disease-free (P = 0.004) and overall survival (P = 0.007). In addition we show that for disease-free survival the prognostic power of this genetic alteration is second only to axillary lymph node involvement (3.1 vs 6.3 relative risk), and is a better predictor than the mutational status of TP53 (1.6 relative risk). Our results are further evidence of the presence, within the region, of at least a second tumour suppressor gene distal to TP53, that might be targeted by deletions.

Alleles↗

PubMed1998

Somatic mutations that contribute to breast cancer.

Cytogenetic and molecular analyses of primary sporadic human breast carcinomas have documented at least 12 different chromosome arms affected by loss of heterozygosity (LOH). This has been taken as evidence for the presence of putative tumour suppressor genes in the remaining allele within the affected regions. We have previously identified three regions on chromosome 17q that are affected by LOH in primary human breast tumours. A physical map of one of these regions (17q21) has been prepared. The putative target gene appears to be located between the D17S846 and D17S746 loci. We are currently determining whether either of two genes located in this region is the target for LOH. The mouse mammary tumour model system provides an approach for identifying genes which, when activated or inactivated by mouse mammary tumour virus (MMTV) integration, contribute to specific stages of mammary tumorigenesis. Using this approach we have identified two genes, designated NOTCH4/INT3 and INT6 respectively. Interruption of NOTCH4/INT3 by MMTV represents a gain-of-function mutation that has profound consequences for mammary gland development and tumorigenesis. INT6 was found to be interrupted by an integrated MMTV genome in a mammary hyperplastic outgrowth line and two independent mammary tumours. In each case the transcriptional orientation of the viral genome was opposite to that of INT6. The rearranged allele was expressed as a truncated chimaeric RNA species composed of INT6 coding sequences, intron sequences and MMTV sequences. Since the non-rearranged allele contained no mutations, we conclude that MMTV integration into INT6 causes a dominant-negative mutation or biologically activates its function. The nucleotide sequence of INT6 is unrelated to any of the known genes in the GenBank database, but is evolutionarily highly conserved.

Animals↗

PubMed1997

The chromosome location of the human homolog of the mouse mammary tumor-associated gene INT6 and its status in human breast carcinomas.

The INT6 gene is a common integration site for the mouse mammary tumor virus in mouse mammary tumors. We have determined that the human homolog of INT6 is located on chromosome region 8q22-q23. A processed INT6 pseudogene is located on chromosome 6q. INT6 is composed of 13 exons that span 45 kb of genomic DNA. The deduced amino acid sequence of the gene product is identical to the mouse protein and contains three potential translation start signals. We have examined 100 primary breast carcinoma DNAs for evidence of genetic alteration affecting INT6. Loss of heterozyosity (LOH) was detected in 11 of 39 (28%) of the tumor samples informative for a polymorphic sequence in intron 7 of INT6. Since single-strand conformation and hybrid mismatch analysis of the remaining allele in these tumor DNAs failed to detect any mutations, we conclude that the target gene for LOH must be closely linked to INT6.

Animals↗

PubMed1997

The mouse mammary tumor associated gene INT3 is a unique member of the NOTCH gene family (NOTCH4).

The INT3 gene is frequently rearranged in mouse mammary tumor virus (MMTV)-induced mammary tumors of the CzechII mouse strain. We have completed the nucleotide sequence of the normal 6.5 Kb INT3 RNA and defined the intron/exon boundaries of the gene. The open reading frame of INT3 RNA should encode a 200 kd protein which shares 60% homology with the mouse homologue of Drosophila NOTCH. INT3 is unique among other members of the NOTCH family by containing 29 instead of 36 EGF-like repeats in the extracellular domain of the gene product. Five novel EGF-like repeats have been created as consequence of apparent small deletions which have occurred within the coding region for the extracellular domain during evolution. Nucleotide sequence analysis of host-viral junction fragments from nine independent MMTV-induced mammary tumors containing a rearranged INT3 gene reveals that all of the integration events occur within a 174 bp region 3' of the sequences encoding the LIN12 repeats in the INT3 extracellular domain and 5' of the sequences encoding the transmembrane domain. Therefore, the only tumorigenic INT3 mutations resulting from MMTV proviral insertions are those which results in the expression of the intracellular domain. This strongly suggests that MMTV-induced activation of INT3 is manifest in the absence of the regulatory action of the extracellular domain, including the LIN12 repeat sequences, leaving the expressed intracellular domain constitutively free to function in its role in mammary tumorigenesis.

Amino Acid Sequence↗

PubMed1997

Characterization of the INT6 mammary tumor gene product.

INT6 is a unique gene, highly conserved throughout evolution and associated with mammary tumorigenesis in the mouse. Although it is expressed in all adult tissues of the mouse and early in embryonic development, its function is unknown. To study the normal distribution and the potential function of the Int6 gene products, we produced antibodies against synthetic peptides specific for the Int6 protein. Western blot and immunoprecipitation analysis demonstrated a 43-kD major gene product that is localized in the cytosolic fraction of mammary cell homogenates. This latter observation is supported by immunoperoxidase analysis, which shows a strong staining anti-Int6 peptide in the perinuclear region of the HC11 mammary epithelial cell line, suggesting a possible localization in the Golgi apparatus. Further immunocytochemical studies in the mouse embryo show that Int6 expression is prevalent in migrating neural crest cells, in the notochord, and in condensing cartilage between 9.5 and 14.5 days of development. In these embryonic tissues, Int6 staining co-localizes with the staining of ricinus lectin, and giantin, proteins that are specifically associated with the Golgi apparatus. The restricted expression of the protein within the Golgi apparatus and its strong conservation throughout evolution suggest that Int6 may perform an essential cellular function.

Amino Acid Sequence↗

PubMed1996

Expression of a truncated Int3 gene in developing secretory mammary epithelium specifically retards lobular differentiation resulting in tumorigenesis.

Insertional mutation of the Int3 gene, a member of the Notch gene family, is frequently associated with primary mouse mammary tumors induced by the mouse mammary tumor virus (MMTV). A major consequence of these mutations is the production of a shortened 2.4-kb tumor specific Int3 RNA transcript that encodes the entire intracellular domain of the Int3 protein. Previous studies have demonstrated that mammary gland development and function was severely impaired in transgenic mice expressing the truncated Int3 gene product from the MMTV viral promoter. Both mammary ductal growth and secretory lobule development were curtailed in these mice. These results were attributed to a gain of function modification of the Int3 gene, which led to a restriction of cell fate selection in the affected mammary epithelial cells. To confirm and extend these findings, truncated Int3 was expressed from the whey acidic protein (WAP) promoter, the activity of which, unlike that of the MMTV long terminal repeat, is restricted to the secretory mammary epithelial population. In transgenic mice carrying the WAP/Int3 construct, mammary ductal growth was unaffected in virgin females, but growth and differentiation of secretory lobules during gestation was profoundly inhibited. Coincidental with the block in lobular secretory differentiation, mammary dysplasia and tumorigenesis occurred in all breeding females by 25 weeks of age. In nonbreeding WAP/Int3 females, mammary tumor incidence also reached 100%, but only after 70 weeks. The WAP/Int3 mammary tumors were highly malignant, and most tumor-bearing females, irrespective of breeding history, developed metastatic lung lesions. These results suggest that WAP promotor-targeted Int3 function is associated with mammary secretory cell differentiation and maintenance in this transgenic model. Consistent with the conclusion that WAP-driven truncated Int3 expression influenced only lobular differentiation and not ductal growth and extension during mammary gland development, transplants of WAP/Int3 gland into nontransgenic mammary fat pads produced complete mammary ductal outgrowths in virgin FVB/N mice but failed to develop secretory lobules when the females were impregnated.

Animals↗

PubMed1996

Detection of amphiregulin and Cripto-1 in mammary tumors from transgenic mice.

Epidermal growth factor family members are widely expressed in human breast cancer and are thought to play an important dual role in mammary gland development and tumorigenesis. Overexpression of two relatively new members of this family, amphiregulin (AR) and Cripto-1 (CR-1), has been previously shown to transform immortalized human and mouse mammary epithelial cells. Here, we extend these results and address the disregulated expression of AR and CR-1 in many types of transgenic neoplastic mouse mammary tissues. Transgenic mouse strains overexpressing the oncogenes transforming growth factor-alpha, neu, int-3, polyoma virus middle T antigen, and simian virus 40 large T antigen have been previously shown to develop spontaneous mammary neoplasia. These models were each examined for mammary-tumor expression of AR and CR-1 by reverse transcription-polymerase chain reaction, western blot, and immunocytochemical analyses. Mammary tumors from each source expressed AR and CR-1. Western blot analysis revealed that, in all mammary tumors, AR and CR-1 protein species were processed differently than in virgin and lactating mouse mammary tissue. In addition, immunohistochemical detection of AR and CR-1 in tumor tissue revealed different patterns of growth-factor localization in different types of transgenic mouse mammary-derived tumors. These findings are consistent with the possibility of widespread roles for AR and CR-1 in the promotion and/or progression stages of mouse mammary tumorigenesis.

Amphiregulin↗

PubMed1996

MMTV-induced mutations in mouse mammary tumors: their potential relevance to human breast cancer.

In mouse mammary tumor virus (MMTV) infected mice, three identifiable stages of mammary tumorigenesis can be biologically defined: preneoplastic hyperplastic nodules, malignant tumor, and distant metastatic lesions (primarily in the lung). MMTV is a biological carcinogen which induces somatic mutations as consequence of its integration into the host cellular genome. Each stage of mammary tumorigenesis appears to result from the clonal outgrowth of cells containing additional integrated proviral MMTV genomes. This phenomenon has provided the basis for an approach to identify genes which, when affected, may contribute to progression through the different stages of mammary tumorigenesis. Eight different genes (Wnt1, Wnt3, Wnt10b, Fgf3, Fgf4, Fgf8, Int3, and Int6) have been shown to be genetically altered in multiple mammary tumors as a consequence of MMTV integration. Although the significance of the human homologs of these genes as targets for somatic mutation during human breast carcinogenesis is only now being explored, it is clear that this work has led to a new appreciation of the complexity of the genetic circuitry that is involved in the control of normal mammary gland growth and development. It seems likely that some of the mutations induced by MMTV, and the signaling pathways in which these target genes take part, will be relevant to the progression from preneoplastic lesions to distant metastasis in human breast cancer.

Animals↗

PubMed1996

Multiple regions of chromosome 6q affected by loss of heterozygosity in primary human breast carcinomas.

A total of 80 primary human breast carcinoma DNAs were analysed for loss of heterozygosity (LOH) on the long arm of chromosome 6, using microsatellite markers whose location has been defined physically and by linkage analysis. Loss of heterozygosity was observed in 38 of 80 (48%) tumours that were informative for at least one locus. The analysis revealed partial or interstitial deletions of chromosome 6q. Detailed mapping of chromosome 6q in these tumour DNAs identified two and perhaps three commonly deleted regions. One of these is located between markers D6S251 and D6S252 (6q14-q16.2), another between D6S268 and D6S261 (6q16.3-q23) and a third between D6S287 and D6S270 (6q22.3-q23.1).

Base Sequence↗