Biomedical subjects

Raju Kucherlapati

Publications and source records attributed to Raju Kucherlapati.

18 recordsLinked to original sources

PubMed2004

An Msh2 point mutation uncouples DNA mismatch repair and apoptosis.

Mutations in the human DNA mismatch repair gene MSH2 are associated with hereditary nonpolyposis colorectal cancer as well as a significant proportion of sporadic colorectal cancer. The inactivation of MSH2 results in the accumulation of somatic mutations in the genome of tumor cells and resistance to the genotoxic effects of a variety of chemotherapeutic agents. Here we show that the DNA repair and DNA damage-induced apoptosis functions of Msh2 can be uncoupled using mice that carry the G674A missense mutation in the conserved ATPase domain. As a consequence, although Msh2(G674A) homozygous mutant mice are highly tumor prone, the onset of tumorigenesis is delayed as compared with Msh2-null mice. In addition, tumors that carry the mutant allele remain responsive to treatment with a chemotherapeutic agent. Our results indicate that Msh2-mediated apoptosis is an important component of tumor suppression and that certain MSH2 missense mutations can cause mismatch repair deficiency while retaining the signaling functions that confer sensitivity to chemotherapeutic agents.

Alanine↗

PubMed2003

Hierarchical model of gene regulation by transforming growth factor beta.

Transforming growth factor betas (TGF-betas) regulate key aspects of embryonic development and major human diseases. Although Smad2, Smad3, and extracellular signal-regulated kinase (ERK) mitogen-activated protein kinases (MAPKs) have been proposed as key mediators in TGF-beta signaling, their functional specificities and interactivity in controlling transcriptional programs in different cell types and (patho)physiological contexts are not known. We investigated expression profiles of genes controlled by TGF-beta in fibroblasts with ablations of Smad2, Smad3, and ERK MAPK. Our results suggest that Smad3 is the essential mediator of TGF-beta signaling and directly activates genes encoding regulators of transcription and signal transducers through Smad3/Smad4 DNA-binding motif repeats that are characteristic for immediate-early target genes of TGF-beta but absent in intermediate target genes. In contrast, Smad2 and ERK predominantly transmodulated regulation of both immediate-early and intermediate genes by TGF-beta/Smad3. These results suggest a previously uncharacterized hierarchical model of gene regulation by TGF-beta in which TGF-beta causes direct activation by Smad3 of cascades of regulators of transcription and signaling that are transmodulated by Smad2 and/or ERK.

Animals↗

PubMed2003

A panel of repeat markers for detection of microsatellite instability in murine tumors.

A substantial fraction of human malignancies lack functional DNA mismatch repair (MMR), accumulate mutations at high frequency, and exhibit microsatellite instability (MSI). In order to distinguish between MMR-competent and MMR-deficient malignancies, a consensus panel of microsatellite repeats has been adopted for MSI analysis of human tumors. There is no reference panel of repeats for MSI typing of murine malignancies. In this study, we present six new microsatellite repeat markers for MSI typing of mouse tumors. Analysis of polymerase chain reaction (PCR)-amplified tumor DNA from MMR-deficient and -proficient mice on denaturing polyacrylamide gels revealed that the new panel of microsatellites was more sensitive in detecting MSI than six commonly used CA(n) repeats. Using the new set of microsatellite markers, we demonstrated the presence of MSI in endometrial carcinoma and cancer precursors from diethylstilbestrol (DES)-treated mice, pointing to a possible role for loss of MMR in hormonally promoted endometrial tumorigenesis.

Adaptor Proteins, Signal Transducing↗

PubMed2003

Spontaneous aberrant crypt foci in Apc1638N mice with a mutant Apc allele.

The Apc1638N/+ mouse has a chain-terminating mutation in one allele of the adenomatous polyposis coli (Apc) gene that is similar to most mutations observed in the human familial adenomatous polyposis syndrome. Aberrant crypt foci (ACF), the earliest identified neoplastic lesions in the colon, are morphologically abnormal structures that are identifiedmicroscopically in the grossly normal colonic mucosas of rodents treated with colon carcinogens and of human patients. The colons and cecums of 62 Apc1638N/+ mice were evaluated for the spontaneous occurrence of ACF and tumors. Both male and female mice were killed at different times between 5 and 28 weeks of age. Wild-type littermates, ie, Apc(+/+) mice, at 22 to 26 weeks of age served as controls. ACF were identified in 97% of the Apc1638N/+ mice starting at 5 weeks of age and not in any wild-type littermates. Although the number of ACF increased with age (P < 0.0001), the average number of crypts per focus of the ACF did not increase significantly. In addition, wild-type Apc protein was detected by immunohistochemistry in all 22 ACF evaluated. Together these data suggest that heterozygous loss of Apc may be sufficient to initiate ACF in these mice and that these mice may be suitable models to study the interaction of environmental factors with an inherited mutation of the Apc gene that is associated with colon cancer.

Adenomatous Polyposis Coli↗

PubMed2003

DNA mismatch repair protein Msh6 is required for optimal levels of ultraviolet-B-induced apoptosis in primary mouse fibroblasts.

Recent data support a role for DNA mismatch repair in the cellular response to some forms of exogenous DNA damage beyond that of DNA repair; cells with defective DNA mismatch repair have partial or complete failure to undergo apoptosis and/or G2M arrest following specific types of damage. We propose that the DNA mismatch repair Msh2/Msh6 heterodimer, responsible for the detection of DNA damage, promotes apoptosis in normal cells, thus protecting mammals from ultraviolet-induced malignant transformation. Using primary mouse embryonic fibroblasts derived from Msh6+/+ and Msh6-/- mice, we compare the response of DNA-mismatch repair-proficient and -deficient cells to ultraviolet B radiation. In the wild-type mouse embryonic fibroblasts, ultraviolet-B-induced increases in Msh6 protein levels were not dependent on p53. Msh6-/- mouse embryonic fibroblasts were significantly less sensitive to the cytotoxic effects of ultraviolet B radiation. Further comparison of the Msh6+/+ and Msh6-/- mouse embryonic fibroblasts revealed that Msh6-/- mouse embryonic fibroblasts undergo significantly less apoptosis following ultraviolet B irradiation, thus indicating that ultraviolet-B-induced apoptosis is partially Msh6 dependent. These data support a role for Msh6 in protective cellular responses of primary cells to ultraviolet-B-induced mutagenesis and, hence, the prevention of skin cancer.

Animals↗

PubMed2003

Differential gene expression following early renal ischemia/reperfusion.

BACKGROUND: Acute renal failure from ischemia/reperfusion injury is associated with tubule cell apoptosis, the molecular mechanisms of which remain under active investigation. The purpose of this study was to identify apoptosis-related genes that are differentially expressed in the early periods following renal ischemia. METHODS: Mice underwent unilateral renal artery clamping for 45 minutes and were sacrificed at 0, 3, 12, or 24 hours of reperfusion. Tubule cell apoptosis was confirmed by DNA laddering and terminal deoxynucleotidyl transferase-mediated uridine triphosphate nick end labeling (TUNEL) assay. We employed cDNA microarrays to define global changes in renal gene expression. Semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) and immunohistochemistry were used as confirmatory tools. RESULTS: By microarray analysis, we identified consistent patterns of altered gene expression, including transcription factors, growth factors, signal transduction molecules, and apoptotic factors. Prominent among the last category included FADD, DAXX, BAD, BAK, and p53. Up-regulation of these proapoptotic genes was confirmed by semiquantitative RT-PCR and immunohistochemistry. CONCLUSION: The results indicate that apoptosis may represent an important mechanism for the early loss of tubule cells following ischemia/reperfusion injury. Both the death receptor-dependent (FADD-DAXX) and mitochondrial (BAD-BAK) pathways are activated. The results also provide a molecular basis for the previous findings that significant intrarenal mechanisms exist to enable tubule cell repair and regeneration, as evidenced by the up-regulation of genes such as growth, proliferation, transcription, and cytoskeletal factors.

Animals↗

PubMed2003

Regional response leading to tumorigenesis after sulindac in small and large intestine of mice with Apc mutations.

Sulindac and other NSAIDs have been widely studied as potential chemopreventive agents for colon cancer. Short-term studies have shown adenomatous polyps to regress in patients with familial adenomatous polyposis (FAP). In this study the effect of sulindac on cancer as an endpoint was evaluated in ApcMin mice, a preclinical model of FAP with an Apc mutation in codon 850 that leads to gastrointestinal adenomas and carcinomas. Three groups of mice were studied all of which were fed AIN-76A diet: one group was fed AIN-76A diet alone, a second group received sulindac 200 p.p.m. premixed in the diet and a third group received sulindac 180 p.p.m. added in drinking water. ApcMin mice were killed 9 weeks after feeding was initiated. Mice receiving sulindac developed fewer tumors in the intestine overall; the major decrease in tumor development after sulindac was seen in the small intestine regardless of route of administration. In the large intestine, however, sulindac significantly increased the incidence, multiplicity and volume of tumors in the colon of ApcMin mice, a regional response to sulindac differing from previous reports. Quantitative measurements of apoptosis, Bax and Bcl-xL protein expression in the ApcMin mice revealed the ratio of Bax/Bcl-xL expression and apoptosis increased in the small intestine but decreased in the cecum, consistent with the regional tumorigenesis observed after sulindac. These findings thus suggest involvement of Bax and apoptosis in tumors developing after sulindac treatment in this mouse model.

Animals↗

PubMed2003

Rb and N-ras function together to control differentiation in the mouse.

The product of the retinoblastoma tumor suppressor gene (Rb) can control cell proliferation and promote differentiation. Murine embryos nullizygous for Rb die midgestation with defects in cell cycle regulation, control of apoptosis, and terminal differentiation of several tissues, including skeletal muscle, nervous system, and lens. Previous cell culture-based experiments have suggested that the retinoblastoma protein (pRb) and Ras operate in a common pathway to control cellular differentiation. Here we have tested the hypothesis that the proto-oncogene N-ras participates in Rb-dependent regulation of differentiation by generating and characterizing murine embryos deficient in both N-ras and Rb. We show that deletion of N-ras rescues a unique subset of the developmental defects associated with nullizygosity of Rb, resulting in a significant extension of life span. Rb(-/-); N-ras(-/-) skeletal muscle has normal fiber density, myotube length and thickness, in contrast to Rb-deficient embryos. Additionally, Rb(-/-); N-ras(-/-) muscle shows a restoration in the expression of the late muscle-specific gene MCK, and this correlates with a significant potentiation of MyoD transcriptional activity in Rb(-/-); N-ras(-/-), compared to Rb(-/-) myoblasts in culture. The improved differentiation of skeletal muscle in Rb(-/-); N-ras(-/-) embryos occurs despite evidence of deregulated proliferation and apoptosis, as seen in Rb-deficient animals. Our findings suggest that the control of differentiation and proliferation by Rb are genetically separable.

Animals↗

PubMed2002

The role of neural crest during cardiac development in a mouse model of DiGeorge syndrome.

The velo-cardio-facial syndrome (VCFS)/DiGeorge syndrome (DGS) is a genetic disorder characterized by phenotypic abnormalities of the derivatives of the pharyngeal arches, including cardiac outflow tract defects. Neural crest cells play a major role in the development of the pharyngeal arches, and defects in these cells are likely responsible for the syndrome. Most patients are hemizygous for a 1.5- to 3.0-Mb region of 22q11, that is suspected to be critical for normal pharyngeal arch development. Mice hemizygous for a 1.5-Mb homologous region of chromosome 16 (Lgdel/+) exhibit conotruncal cardiac defects similar to those seen in affected VCFS/DGS patients. To investigate the role of Lgdel genes in neural crest development, we fate mapped neural crest cells in Lgdel/+ mice and we performed hemizygous neural crest-specific inactivation of Lgdel. Hemizygosity of the Lgdel region does not eliminate cardiac neural crest migration to the forming aortic arches. However, neural crest cells do not differentiate appropriately into smooth muscle in both fourth and sixth aortic arches and the affected aortic arch segments develop abnormally. Tissue-specific hemizygous inactivation of Lgdel genes in neural crest results in normal cardiovascular development. Based on our studies, we propose that Lgdel genes are required for the expression of soluble signals that regulate neural crest cell differentiation.

Animals↗

PubMed2002

APC-dependent suppression of colon carcinogenesis by PPARgamma.

Activation of PPARgamma by synthetic ligands, such as thiazolidinediones, stimulates adipogenesis and improves insulin sensitivity. Although thiazolidinediones represent a major therapy for type 2 diabetes, conflicting studies showing that these agents can increase or decrease colonic tumors in mice have raised concerns about the role of PPARgamma in colon cancer. To analyze critically the role of this receptor, we have used mice heterozygous for Ppargamma with both chemical and genetic models of this malignancy. Heterozygous loss of PPARgamma causes an increase in beta-catenin levels and a greater incidence of colon cancer when animals are treated with azoxymethane. However, mice with preexisting damage to Apc, a regulator of beta-catenin, develop tumors in a manner insensitive to the status of PPARgamma. These data show that PPARgamma can suppress beta-catenin levels and colon carcinogenesis but only before damage to the APC/beta-catenin pathway. This finding suggests a potentially important use for PPARgamma ligands as chemopreventative agents in colon cancer.

Animals↗

PubMed2002

Haploinsufficiency of Flap endonuclease (Fen1) leads to rapid tumor progression.

Flap endonuclease (Fen1) is required for DNA replication and repair, and defects in the gene encoding Fen1 cause increased accumulation of mutations and genome rearrangements. Because mutations in some genes involved in these processes cause cancer predisposition, we investigated the possibility that Fen1 may function in tumorigenesis of the gastrointestinal tract. Using gene knockout approaches, we introduced a null mutation into murine Fen1. Mice homozygous for the Fen1 mutation were not obtained, suggesting absence of Fen1 expression leads to embryonic lethality. Most Fen1 heterozygous animals appear normal. However, when combined with a mutation in the adenomatous polyposis coli (Apc) gene, double heterozygous animals have increased numbers of adenocarcinomas and decreased survival. The tumors from these mice show microsatellite instability. Because one copy of the Fen1 gene remained intact in tumors, Fen1 haploinsufficiency appears to lead to rapid progression of cancer.

Adenocarcinoma↗

PubMed2002

Microarray-based copy number and expression profiling in dedifferentiated and pleomorphic liposarcoma.

Sixteen dedifferentiated and pleomorphic liposarcomas were analyzed by comparative genomic hybridization (CGH) to genomic microarrays (matrix-CGH), cDNA-derived microarrays for expression profiling, and by quantitative PCR. Matrix-CGH revealed copy number gains of numerous oncogenes, i.e., CCND1, MDM2, GLI, CDK4, MYB, ESR1, and AIB1, several of which correlate with a high level of transcripts from the respective gene. In addition, a number of genes were found differentially expressed in dedifferentiated and pleomorphic liposarcomas. Application of dedicated clustering algorithms revealed that both tumor subtypes are clearly separated by the genomic profiles but only with a lesser power by the expression profiles. Using a support vector machine, a subset of five clones was identified as "class discriminators." Thus, for the distinction of these types of liposarcomas, genomic profiling appears to be more advantageous than RNA expression analysis.

Algorithms↗

PubMed2002

Initiating oncogenic event determines gene-expression patterns of human breast cancer models.

Molecular expression profiling of tumors initiated by transgenic overexpression of c-myc, c-neu, c-ha-ras, polyoma middle T antigen (PyMT) or simian virus 40 T/t antigen (T-ag) targeted to the mouse mammary gland have identified both common and oncogene-specific events associated with tumor formation and progression. The tumors shared great similarities in their gene-expression profiles as compared with the normal mammary gland with an induction of cell-cycle regulators, metabolic regulators, zinc finger proteins, and protein tyrosine phosphatases, along with the suppression of some protein tyrosine kinases. Selection and hierarchical clustering of the most variant genes, however, resulted in separating the mouse models into three groups with distinct oncogene-specific patterns of gene expression. Such an identification of targets specified by particular oncogenes may facilitate development of lesion-specific therapeutics and preclinical testing. Moreover, similarities in gene expression between human breast cancers and the mouse models have been identified, thus providing an important component for the validation of transgenic mammary cancer models.

Animals↗

PubMed2002

Colorectal cancer in mice genetically deficient in the mucin Muc2.

The gastrointestinal tract is lined by a layer of mucus comprised of highly glycosylated proteins called mucins. To evaluate the importance of mucin in intestinal carcinogenesis, we constructed mice genetically deficient in Muc2, the most abundant secreted gastrointestinal mucin. Muc2-/- mice displayed aberrant intestinal crypt morphology and altered cell maturation and migration. Most notably, the mice frequently developed adenomas in the small intestine that progressed to invasive adenocarcinoma, as well as rectal tumors. Thus, Muc2 is involved in the suppression of colorectal cancer.

Adenocarcinoma↗

PubMed2002

Mechanism for the learning deficits in a mouse model of neurofibromatosis type 1.

Neurofibromatosis type I (NF1) is one of the most common single-gene disorders that causes learning deficits in humans. Mice carrying a heterozygous null mutation of the Nfl gene (Nfl(+/-) show important features of the learning deficits associated with NF1 (ref. 2). Although neurofibromin has several known properties and functions, including Ras GTPase-activating protein activity, adenylyl cyclase modulation and microtubule binding, it is unclear which of these are essential for learning in mice and humans. Here we show that the learning deficits of Nf1(+/-) mice can be rescued by genetic and pharmacological manipulations that decrease Ras function. We also show that the Nf1(+/-) mice have increased GABA (gamma-amino butyric acid)-mediated inhibition and specific deficits in long-term potentiation, both of which can be reversed by decreasing Ras function. Our results indicate that the learning deficits associated with NF1 may be caused by excessive Ras activity, which leads to impairments in long-term potentiation caused by increased GABA-mediated inhibition. Our findings have implications for the development of treatments for learning deficits associated with NF1.

Animals↗

PubMed2002

DNA mismatch repair deficiency accelerates endometrial tumorigenesis in Pten heterozygous mice.

PTEN mutation and microsatellite instability are two of the most common genetic alterations in uterine endometrioid carcinoma. Furthermore, previous studies have suggested an association between the two alterations, however the basis and consequence of the association is not understood. Recently it has been shown that 100% of female Pten(+/-) mice develop complex atypical hyperplasia by 32 weeks of age that progresses to endometrial carcinoma in approximately 20 to 25% of mice at 40 weeks. In an attempt to expand this mouse model of endometrial tumorigenesis and to further our understanding of the association betweenPten mutations and DNA mismatch repair deficiency, we generated Ptenheterozygous, Mlh1-null (mismatch repair deficient) mice. Significantly, the majority ofPten(+/-)/Mlh1(-/-)mice developed polypoid lesions in the endometrium at 6 to 9 weeks of age. By 14 to 18 weeks, all of the double-mutant mice had lesions histologically similar to those seen inPten(+/-) mice, and two of them exhibited invasive disease. Moreover, the frequency of loss of the wild-type Pten allele in the double-mutant mice at 14 to 18 weeks was similar to that seen in lesions from 40-week-old Pten(+/-) mice. Taken together, our results indicate that DNA mismatch repair deficiency can accelerate endometrial tumorigenesis inPten heterozygous mice and suggests that loss of the wild-type Pten allele is involved in the development/progression of tumors in this setting.

Adaptor Proteins, Signal Transducing↗

PubMed2002

Mouse models for human DNA mismatch-repair gene defects.

The mammalian DNA mismatch-repair genes belong to a family of genes that comprise several homologs of the Escherichia coli mutS and mutL genes. The observation that mutations in the two human repair genes MSH2 and MLH1 are responsible for hereditary nonpolyposis colorectal cancer, as well as a significant number of sporadic colorectal cancers, raises several questions about the role of these proteins and their family members in the initiation and progression of colorectal cancer. To address these questions, mice with inactivating mutations in all the known mutS and mutL homologs have been generated. The development of these mouse lines has permitted the systematic analysis of the role of each gene in the repair process and has underscored their significance in mutation avoidance and cancer susceptibility. These analyses were critical for our understanding of the function of these genes at the organismal level and also revealed an essential role for some of the DNA mismatch-repair genes in mammalian meiosis.

Adenosine Triphosphatases↗

PubMedDate not supplied

Cyclin D1 expression in the intestinal mucosa and tumors of Apc1638N mice.

Altered expression of cyclin D1 contributes to the development of several types of cancer, including colorectal cancer. This study examined cyclin D1 expression in 32 intestinal tumors in different stages of tumorigenesis in Apc1638N mice, a mouse model for human familial adenomatous polyposis (FAP). Three morphological patterns of expression of cyclin D1 in intestinal epithelial cells were found: nuclear, punctate-cytoplasmic and fine granular cytoplasmic. The nuclear pattern of cyclin D1 was detected in all of the tumors, including adenomas (n = 18) and adenocarcinomas (n = 14); this pattern was found predominantly in the tubular region of the tumors and in flat mucosa adjacent to a subset of the tumors (67% of adenomas and 57% of carcinomas). The punctate-cytoplasmic pattern of cyclin D1 expression was found in all adenocarcinomas and a majority of adenomas (80%), mainly in invasive and villous areas of the tumors; it was not found in normal flat adjacent mucosa suggesting that this pattern and altered cytoplasmic/nuclear expression were associated with tumor progression. Fine cytoplasmic granules were located in normal duodenum in the basal portion of the crypts and in colon in epithelial cells at the surface of the colonic crypts; in both duodenum and colon the number of cells with fine cytoplasmic granules significantly increased after feeding a Western-style diet. These altered patterns of expression of cyclin D1 may provide useful biomarkers of abnormal cell development for studies of tumorigenesis and the effects of chemopreventive agents.

Adenomatous Polyposis Coli↗