Biomedical subjects

J C Barrett

Publications and source records attributed to J C Barrett.

At least 91 records · Page 5Linked to original sources

PubMed1999

Frequent loss of KAI1 expression in squamous and lymphoid neoplasms. An immunohistochemical study of archival tissues.

The metastasis suppressor gene KAI1 was identified by its ability to inhibit the formation of pulmonary metastases in experimental models for prostatic carcinoma. Down-regulation of this gene may be correlated with the invasive phenotype in melanomas and colon and bladder carcinomas and with the metastatic phenotype in carcinomas of the lung, breast, prostate, and pancreas. The goal of our study was to establish an immunohistochemical method to detect KAI1 expression in archival tissues. Using cell lines with known KAI1 levels and paraffin-embedded KAI1 positive tissues as controls, we observed strong membrane staining in lymphoid follicular centers and squamous epithelia. We then demonstrated the utility of our assay by studying KAI1 expression in 34 lymphoid and 57 squamous lesions. All eight reactive lymph nodes were KAI1 positive. In contrast, three of 13 follicular small cleaved and five of 13 diffuse large cell lymphomas were KAI1 negative. Seventy-nine percent (37 of 47) of invasive squamous cell carcinomas from the lung (n = 15), head and neck (n = 18), and cervix (n = 14) showed extensive KAI1 down-regulation. Loss of KAI1 expression was also found in a subset of 10 high-grade cervical dysplasias. Our data show that (i) immunohistochemistry is a suitable technique for evaluating KAI1 expression in archival tissues; (ii) KAI1 was not expressed in a subset of both low-grade and high-grade lymphomas; and (iii) there was extensive down-regulation of KAI1 in squamous cell carcinomas, suggestive of an important role of the gene in the suppression of invasion in these malignancies.

Animals↗

PubMed1999

Increased insulin sensitivity and hypoglycaemia in mice lacking the p85 alpha subunit of phosphoinositide 3-kinase.

The hallmark of type 2 diabetes, the most common metabolic disorder, is a defect in insulin-stimulated glucose transport in peripheral tissues. Although a role for phosphoinositide-3-kinase (PI3K) activity in insulin-stimulated glucose transport and glucose transporter isoform 4 (Glut4) translocation has been suggested in vitro, its role in vivo and the molecular link between activation of PI3K and translocation has not yet been elucidated. To determine the role of PI3K in glucose homeostasis, we generated mice with a targeted disruption of the gene encoding the p85alpha regulatory subunit of PI3K (Pik3r1; refs 3-5). Pik3r1-/- mice showed increased insulin sensitivity and hypoglycaemia due to increased glucose transport in skeletal muscle and adipocytes. Insulin-stimulated PI3K activity associated with insulin receptor substrates (IRSs) was mediated via full-length p85 alpha in wild-type mice, but via the p50 alpha alternative splicing isoform of the same gene in Pik3r1-/- mice. This isoform switch was associated with an increase in insulin-induced generation of phosphatidylinositol(3,4,5)triphosphate (PtdIns(3,4,5)P3) in Pik3r1-/- adipocytes and facilitation of Glut4 translocation from the low-density microsome (LDM) fraction to the plasma membrane (PM). This mechanism seems to be responsible for the phenotype of Pik3r1-/- mice, namely increased glucose transport and hypoglycaemia. Our work provides the first direct evidence that PI3K and its regulatory subunit have a role in glucose homeostasis in vivo.

Animals↗

PubMed1999

The linoleic acid metabolite, 13-HpODE augments the phosphorylation of EGF receptor and SHP-2 leading to their increased association.

In previous studies with Syrian hamster embryo fibroblasts, we found that a specific lipoxygenase metabolite of linoleic acid, 13(S)-hydroperoxyoctadecadienoic acid (HpODE), enhanced epidermal growth factor (EGF) signal transduction in a tumor suppressor gene plus phenotype (supB+); with a diminished response to 13(S)-HpODE in a tumor suppressor gene minus phenotype (supB-). This differential response was attributed to differences in the rate of EGF receptor (EGFR) dephosphorylation. To further define the molecular basis for these observations, in this report we examine the interaction of phosphorylated EGFR with the SH2 domain-containing protein tyrosine phosphatase, SHP-2, a positive modulator of EGF dependent cell growth. SHP-2 associated with phosphorylated EGFR to a greater extent in supB+ cells when compared to supB-. This differential association could not be accounted for by differences between suppressor gene phenotypes in SHP-2 protein level or mutations in the molecular sequence. The addition of 13(S)-HpODE stimulated a concentration-dependent increase in EGF-dependent phosphorylation of SHP-2 and its association with EGFR. A more dramatic response was observed in the supB+ cells. Differences in SHP-2 interaction with EGFR may account, in part, for phenotypic differences in the growth rates and responsiveness to EGF between the supB+ and supB- cells. EGFR-SHP-2 association appears to play an important role in the regulation of EGFR signal transduction.

Animals↗

PubMed1999

The oncostatic action of melatonin in an ovarian carcinoma cell line.

Melatonin is reported to reduce proliferation in many cell types, but the effect is small and the results are inconsistent. Information on the mechanism by which melatonin exerts its antiproliferative effects might provide insight into the variability of the response. In an ovarian adenocarcinoma cell line (BG-1), we find that melatonin at concentrations of 10(-9)-10(-7) M caused a 20-25% reduction in cell number. Melatonin also resulted in a similar reduction in [3H]-thymidine incorporation with no significant increase in cell death as measured by trypan blue incorporation. The Kd for melatonin reduction in cell number was approximately 5 x 10(-10) M. Melatonin ML2 receptors have a Kd for melatonin binding in the low nM range and are linked to the production of the calcium mobilizing agent inositol-1,4,5-trisphosphate (IP3). To investigate whether melatonin signaling involves an increase in cytosolic-free calcium. BG-1 cells were loaded with the calcium sensitive indicator, fura-2. Acute addition of melatonin (10(-5)-10(-9) M) did not alter cytosolic calcium. Addition of the putative nuclear receptor agonist CGP52608 caused a dose-dependent inhibition of cell number with a Kd of approximately 2 x 10(-9) M. Addition of CGP52608 caused a similar reduction in [3H]-thymidine incorporation. Neither melatonin (10(-8) M-10(-5) M) nor CGP52608 at concentrations below 10(-7) M induced cell death associated with the inhibition of cell proliferation; however, addition of CGP52608 at a high dose (10(-7) M) caused an increase in cell death, consistent with apoptosis. Growth inhibition by melatonin or CGP52608 did not alter the percentage of cells in G1 versus S/G2/M.

Adenocarcinoma↗

PubMed1999

Identification of a gene that reverses the immortal phenotype of a subset of cells and is a member of a novel family of transcription factor-like genes.

Based on the dominance of cellular senescence over immortality, immortal human cell lines have been assigned to four complementation groups for indefinite division. Human chromosomes carrying senescence genes have been identified, including chromosome 4. We report the cloning and identification of a gene, mortality factor 4 (MORF 4), which induces a senescent-like phenotype in immortal cell lines assigned to complementation group B with concomitant changes in two markers for senescence. MORF 4 is a member of a novel family of genes with transcription factor-like motifs. We present here the sequences of the seven family members, their chromosomal locations, and a partial characterization of the three members that are expressed. Elucidation of the mechanism of action of these genes should enhance our understanding of growth regulation and cellular aging.

Amino Acid Sequence↗

PubMed1999

Functional roles of chromosomes 11 and 17 in the transformation of human breast epithelial cells in vitro.

Genomic alterations in primary breast cancer play a role in the initiation and progression of the disease. We have analyzed the molecular events involved in the initiation and progression of the neoplastic process in an in vitro experimental system. Immortalization of human breast epithelial cells (HBEC) is associated with 3:9 translocation, p53 mutation and microsatellite instability (MSI) of chromosomes 11p13, and 17p. BP1-E cells, derived from the immortalized MCF-10F cells transformed by the carcinogen benzo(a)pyrene (BP), express in vitro growth advantage, anchorage independence, enhanced chemoinvasiveness, loss of ductulogenic capabilities and tumorigenesis in a heterologous host. This neoplastic progression is also associated with mutations and/or amplification of c-H-ras, int-2, c-neu, c-myc and MDM2, MSI at 11q25 and 13q12-q13 and loss of heterozygosity at 17p. In order to test whether chromosomes 11 or 17 play a functional role in the phenotypic expression of transformation of BP1E cells, we utilized microcell-mediated chromosome transfer (MMCT) technique for inserting the corresponding normal chromosomes to these transformed cells. BP1E cells were transfected with PsV2neo plasmid and fused with microcells obtained from the mouse cell line A9, containing a normal chromosome 11 or 17 (A9-11neo and A9-17neo cells, selected in G418 and cloned. Sixteen primary microcell hybrids from each chromosome transfer, designated BP1E-11neo and BP1E-17neo survived selection in G-418 containing medium. A single clone from each group, BP1E-11neo #145 and BP1E-17neo D100, survived subcloning and were utilized for a detailed panel of analyses. The presence of a donor chromosome was confirmed by dual color fluorescence in situ hybridization (FISH), southern blot analysis of the marker vector pSV2neo, and microsatellite polymorphism analysis. The transfer of the normal chromosomes 11 and 17 resulted in a 50% and 90% inhibition of cell growth respectively, and reduced both colony efficiency and colony size. Telomerase activity was significantly reduced only by chromosome 17 insertion, providing a possible explanation for the more significant senescence observed in BP1E-17neo D100 cells. Microsatellite polymorphism analysis revealed that three loci, 11q13-23, 11q23.1, and 11q23.3 (markers D11S911, DRD2, and D11S29) were retained in BP1E-11neo #145 cells, and two, 17q24.2-25.2, 17q25.2 (markers D17S515 and D17S785 were retained in BP1E-17neo D100 cells. We conclude that the specific regions of normal chromosomes 11 and 17 transferred play a functional role in the expression of immortal and transformed phenotypes of HBEC in vitro.

Blotting, Southern↗

PubMed1999

Differential regulation of p21 by p53 and Rb in cellular response to oxidative stress.

Oxidative stress to mammalian cells causes cellular damage and triggers inducible cellular responses leading to cell death by apoptosis. In this paper, we report that p53 was required for programmed cell death induced by oxidative stress in both mouse and human cells and that p53 transactivation was involved in induction of oxidative cell death. Furthermore, we show that p21 was highly responsive to oxidative stress in a p53-dependent manner and that ectopic expression of p21 could increase cellular susceptibility to oxidative stress in the absence of p53. However, p21 was not required for p53-directed oxidative cell death because mouse embryo fibroblasts MEFs lacking p21(p21-/- MEFs) were still susceptible to oxidative cell death. Interestingly, bax, a cell-death mediator regulated by p53, was overexpressed in p21-/- MEFs that underwent cell death by oxidative stress, suggesting a compensation for loss of p21 that may be responsible for the existence of cell-death responses in p21-knockout mouse fibroblasts. Finally, we provide evidence that the retinoblastoma gene product (Rb) is a negative regulator of p21 and a repressor of the cellular apoptotic process. Because p21 is regulated by p53 positively and by Rb negatively, p21 may be a link between p53 and Rb in determining cell fate after oxidative damage.

Animals↗

PubMed1999

Mutations and altered expression of the human cancer genes: what they tell us about causes.

To understand the causes of cancer, it is necessary to elucidate the molecular basis and environmental factors that influence the carcinogenesis process. Cancers are progressive diseases characterized by the accumulation of defects in many different genes. The patterns of mutation of some genes identified in tumours suggest a direct action of chemicals binding to and altering DNA. Other cancer-associated genes may be altered as a consequence of endogenous mutagens, germ-line mutations, spontaneous mutations that occur during cell replication or increased genetic instability in precancerous cells. Recent advances in molecular biology and genetics have provided new tools and concepts for studying the causes of cancer. We know that cancers are caused by a combination of environmental and genetic factors, and the discovery of the molecular alterations that occur at various stages in different tumours is increasing our understanding of these causes. Thus, we are now beginning to discover which genes are involved, how they function normally and in tumour tissues and why cancers develop after a series of genetic and epigenetic changes in certain cells. As data from studies on cancer-associated genes have accrued, the categories of genes and molecular pathways that have been found to play a role in carcinogenesis have also increased. Genes involved in development and other normal cellular processes have been implicated in cancer. These include genes involved in signal transduction, cell cycle control, DNA repair, cell growth and differentiation (growth factors and growth factor receptors), transcriptional regulation, senescence and apoptosis. Genes involved in angiogenesis, immune regulation, cellular responses to stress, motility, adhesion and invasion are also involved, but less is known about their relationship to carcinogenesis, and these processes are not discussed in this review. The diverse nature of these categories of cancer-related genes indicates the variety of processes that must be disrupted in order for tumours to develop. Many of the genes have several functional domains, and the functions of some have only recently been proposed. In this review, we describe some of the major classes of genes implicated in human cancers and some of the major findings on genetic alterations and dysfunction in human tumours. Comparisons are made with certain rodent models.

Animals↗

PubMed1999

Roles for insulin-like growth factor-1 in mediating the anti-carcinogenic effects of caloric restriction.

This paper focuses on the role of insulin-like growth factor-1 (IGF-1) and its associated regulatory apparatus as a key endocrine, autocrine, and paracrine signalling system involved in mediating the anti-carcinogenic activity of dietary restriction. Literature is reviewed showing that the inhibitory action of dietary restriction on carcinogenesis is global and pervasive--it is effective in several laboratory species, for a variety of tumor types, and for both spontaneous tumors and tumors caused by different types of tumor-inducing agents. Evidence is presented showing the IGF-1 pathway responds appropriately to nutritional interventions including diet restriction. Recent evidence points to an obligatory role for the IGF-1 receptor in the establishment and maintenance of the transformed phenotype and reveals that IGF-1 in concert with insulin-like binding protein 3 and p53 is involved in autocrine/paracrine growth signaling pathways as adaptive responses to environmental stimuli. Considered together these works show that the IGF-1 pathway is uniquely poised to influence cellular transformation leading to the malignant phenotype by modulating the balance of cellular proliferation and cell death (apoptosis) in precancerous and cancerous cells and by influencing metastasis of nascent tumors. We evaluated these hypotheses directly using animal models of mononuclear cell leukemia, bladder transitional cell carcinogenesis, and breast cancer. Our studies demonstrate that manipulation of IGF-1 level through dietary intervention influences tumor growth and metastasis. Upregulation of this pathway demonstrated that increased IGF-1 stimulates tumor proliferation, progression and metastasis. Conversely, downregulation of this pathway in vivo as a consequence of dietary restriction results in antitumorigenic activity. We found that the functional disruption of IGF-1R markedly influences breast cancer metastasis in nude mice by suppressing cellular adhesion, invasion, and metastasis of breast cancer cells to the lung, lymph nodes, and lymph vessels. Epidemiological observations and clinical oncology results support the involvement of IGF-1 in carcinogenesis and anticarcinogenesis. This leads to the hypothesis that factors such as IGF-1 which regulate body size and composition may be related to human cancer incidence or prognosis. Additional understanding of this pathway and its interactions with other signaling pathways will advance our ability to develop new interventions towards decreased cancer risk in humans.

Aging↗

PubMed1998

Identification of the rat homologue of KAI1 and its expression in Dunning rat prostate cancers.

BACKGROUND: We previously isolated the human KAI1 gene encoding a transmembrane protein which suppresses metastatic ability in Dunning R3327 AT6.1 rat prostate cancer cells when transfected into these cells. The AT6.1 subline is one of the more aggressive sublines among the Dunning R-3327 system of rat prostate cancers. This raises the issue of whether downregulation of KAI1 expression consistently occurs during the acquisition of high metastatic ability by members of the Dunning system of rat prostate cancers. METHODS: To investigate this possibility, the rat homologue of the KAI1gene was identified, using a combination of cDNA library screening and 5'-RACE and DNA sequencing. Based on this information, a rat-specific cDNA probe was developed and used for Northern blot analysis of KAI1 expression in normal rat tissues and a series of sublines of Dunning R3327 cells that vary widely in their metastatic abilities. RESULTS: The rat KAI1 gene encoded a protein of 266 amino acids which has 77% identity to the human KAI1 protein. In normal tissues, KAI1 is expressed predominantly as a 2.0-kb-sized transcript. Several tissues (e.g., skeletal muscle and prostate) also express a minor 1.8-kb-sized RNA. Northern blot analysis of a series of Dunning sublines demonstrated that all sublines expressed both the 2.0- and 1.8-kb KAI1 RNA transcripts. However, quantitative levels of the 2.0- vs. 1.8-kb KAI1 RNA were variable among sublines. Downregulation of expression of the 2.0-kb KAI1 transcript was statistically correlated with the acquisition of high metastatic ability within this system of prostate cancer sublines. In contrast, the 1.8-kb transcript was upregulated in all of the more aggressive sublines, but this enhanced expression was not specifically correlated with metastatic ability. CONCLUSIONS: These studies demonstrated that downregulation of the 2.0-kb KAI1 mRNA is associated with the acquisition of high metastatic ability by prostate cancer cells.

Amino Acid Sequence↗

PubMed1998

Failure to complement abnormal phenotypes of simian virus 40-transformed Werner syndrome cells by introduction of a normal human chromosome 8.

The Werner syndrome (WS) gene (WRN) was isolated by positional cloning, based on mapping to chromosome 8p12, and the WRN protein was recently shown to encode an active helicase. To examine functional complementation of WS phenotypes by expression of the WRN gene, we introduced a normal human chromosome 8 into a SV40-transformed WS fibroblastoid cell line (WS780) by microcell fusion and studied several cellular phenotypes associated previously with WS cell lines, including cell growth rate, 4-nitroquinoline-1-oxide (4NQO) sensitivity, and spontaneous mutation rate and type of mutation at the hypoxanthine-guanine phosphoribosyl transferase locus. The WRN gene was expressed in two of three microcell hybrids introduced with chromosome 8. We failed to observe a difference between normal and WS cell lines in terms of growth rates and spontaneous mutation rates. However, we found that the WS cell line was highly sensitive to 4NQO-induced cytotoxicity and showed an unusually high proportion of deletion mutations at the hypoxanthine-guanine phosphoribosyl transferase locus as compared to a control cell line, as shown previously. Here, we report that these phenotypes were not corrected by introduction of the WRN gene, although the WRN protein was expressed. Our results suggest that the hypersensitivity to 4NQO and the extensive deletion mutations observed in the WS cell line are caused by a defect that is secondary to the WRN gene mutation, possibly a repair gene defect that controls the phenotypes of hypersensitivity to carcinogen(s) and/or the extensive deletion mutations.

4-Nitroquinoline-1-oxide↗

PubMed1998

Cellular senescence in telomerase-expressing Syrian hamster embryo cells.

We have observed that normal, diploid Syrian hamster embryo cells (SHE) express the enzyme telomerase but undergo senescence at the end of their replicative lifespan. After 20-30 population doublings (pd) these cells cease proliferating, enlarge in size, exhibit a pH 6.0 senescence-associated beta-galactosidase activity, and fail to phosphorylate the RB protein or enter into S-phase after serum stimulation. We have observed that SHE cells express telomerase throughout their replicative lifespan and that the average telomere length does not appear to decrease, remaining at about 23 kb in senescent cells. In addition, individual clones of SHE cells also have telomerase activity and telomeres that do not decrease in length, ruling out the possibility that there is a rare, immortal subpopulation of telomerase-expressing cells that is lost during passaging. Together, these data suggest that SHE cells are likely to senesce by a mechanism that does not involve telomere loss.

Animals↗

PubMed1998

Characterization of distinct human endometrial carcinoma cell lines deficient in mismatch repair that originated from a single tumor.

The role of specific mismatch repair (MMR) gene products was examined by observing several phenotypic end points in two MMR-deficient human endometrial carcinoma cell lines that were originally isolated from the same tumor. The first cell line, HEC-1-A, contains a nonsense mutation in the hPMS2 gene, which results in premature termination and a truncated hPMS2 protein. In addition, HEC-1-A cells carry a splice mutation in the hMSH6 gene and lack wild-type hMSH6 protein. The second cell line, HEC-1-B, possesses the same defective hMSH6 locus. However, HEC-1-B cells are heterozygous at the hPMS2 locus; that is, along with carrying the same nonsense mutation in hPMS2 as in HEC-1-A, HEC-1-B cells also contain a wild-type hPMS2 gene. Initial recognition of mismatches in DNA requires either the hMSH2/hMSH6 or hMSH2/hMSH3 heterodimer, with hPMS2 functioning downstream of damage recognition. Therefore, cells defective in hPMS2 should completely lack MMR (HEC-1-A), whereas cells mutant in hMSH6 only (HEC-1-B) can potentially repair damage via the hMSH2/hMSH3 heterodimer. The data presented here in HEC-1-B cells illustrate (i) the reduction of instability at microsatellite sequences, (ii) a significant decrease in frameshift mutation rate at HPRT, and (iii) the in vitro repair of looped substrates, relative to HEC-1-A cells, illustrating the repair of frameshift intermediates by hMSH2/hMSH3 heterodimer. Furthermore, the role of hMSH2/hMSH3 heterodimer in the repair of base:base mismatches is supported by observing the reduction in base substitution mutation rate at HPRT in HEC-1-B cells (hMSH6-defective but possessing wild-type hPMS2), as compared with HEC-1-A (hMSH6/hPMS2-defective) cells. These data support a critical role for hPMS2 in human MMR, while further defining the role of the hMSH2/hMSH3 heterodimer in maintaining genomic stability in the absence of a wild-type hMSH2/hMSH6 heterodimer.

Base Pair Mismatch↗

PubMed1998

Melatonin attenuates hydrogen peroxide toxicity in MCF7 cells only at pharmacological concentrations.

Melatonin is proposed to be oncostatic in mammary tissue, and one mechanism by which this hormone may elicit its possible oncostatic effect is as an oxygen radical scavenger. Therefore, we examined melatonin's abilities to act as an oxygen radical scavenger at physiological or pharmacological concentrations. Hydrogen peroxide at 400 microM killed 97% of treated MCF7 cells within 8 h, and following melatonin at 10(-5) and 10(-4) M concentrations only 76 and 64% of cells, respectively, were killed by hydrogen peroxide. However, melatonin at lower concentrations (10(-7) M) did not protect MCF7 cells. Moreover, pretreatment with melatonin (10(-5) or 10(-7) M) prior to hydrogen peroxide stress offered no further efficacy, and pretreatment with melatonin followed by the withdrawal of melatonin eliminated its protective effect from hydrogen peroxide toxicity. These findings indicate that melatonin acts directly as an antioxidant and does not stimulate antioxidant defenses in MCF7 cells that protect against hydrogen peroxide. Glutathione levels were examined to substantiate this hypothesis and were not altered by melatonin treatment. In conclusion, melatonin is an excellent oxygen radical scavenger at pharmacological concentrations, but not at physiological concentrations. Thus, loss of melatonin is unlikely to be important in oxidative scavenger mechanisms in human mammary cells.

Antioxidants↗

PubMed1998

The role of hMLH1, hMSH3, and hMSH6 defects in cisplatin and oxaliplatin resistance: correlation with replicative bypass of platinum-DNA adducts.

Defects in mismatch repair are associated with cisplatin resistance, and several mechanisms have been proposed to explain this correlation. It is hypothesized that futile cycles of translesion synthesis past cisplatin-DNA adducts followed by removal of the newly synthesized DNA by an active mismatch repair system may lead to cell death. Thus, resistance to platinum-DNA adducts could arise through loss of the mismatch repair pathway. However, no direct link between mismatch repair status and replicative bypass ability has been reported. In this study, cytotoxicity and steady-state chain elongation assays indicate that hMLH1 or hMSH6 defects result in 1.5-4.8-fold increased cisplatin resistance and 2.5-6-fold increased replicative bypass of cisplatin adducts. Oxaliplatin adducts are not recognized by the mismatch repair complex, and no significant differences in bypass of oxaliplatin adducts in mismatch repair-proficient and -defective cells were found. Defects in hMSH3 did not alter sensitivity to, or replicative bypass of, either cisplatin or oxaliplatin adducts. These observations support the hypothesis that mismatch repair defects in hMutL alpha and hMutS alpha, but not in hMutS beta, contribute to increased net replicative bypass of cisplatin adducts and therefore to drug resistance by preventing futile cycles of translesion synthesis and mismatch correction.

Adaptor Proteins, Signal Transducing↗

PubMed1998

A dominant negative mutant of the insulin-like growth factor-I receptor inhibits the adhesion, invasion, and metastasis of breast cancer.

The 5-year survival rate for women with metastatic breast cancer is only 25-30%; thus, the need to improve treatment is apparent. Overexpression of insulin-like growth factor-I receptor (IGF-IR) correlates with poor prognosis and local recurrence. In this study, we addressed whether functional impairment of IGF-IR affects adhesion, invasion, and metastasis of breast cancer. Impairment of IGF-IR function was achieved by transfecting a dominant negative form of the receptor, termed 486stop, into MDA-MB-435 metastatic breast cancer cells. The protein product of 486stop is secreted extracellularly, resulting in a bystander effect. Cellular adhesion to laminin and collagen was inhibited 94 and 88%, respectively. Furthermore, 486stop inhibited insulin-like growth factor-I-stimulated invasion through collagen IV by 75%. The dominant negative receptor was secreted, as evidenced by the observation that MDA-MB-435 and MDA-MB-231 cells were prevented from binding to laminin by 90% when treated with conditioned medium (CM) from 486stop-transfected cells. CM also inhibited the invasion of MDA-MB-231 cells across collagen IV by 80%. Finally, CM made MDA-MB-231 cells 30% more sensitive to Taxol-induced cell death. Growth in soft agar was suppressed by 486stop, but growth in monolayer was unaffected. When injected into the mammary fat pad, 486stop did not significantly suppress growth of the primary tumor, but metastasis to the lungs, livers, lymph nodes, and lymph vessels was significantly decreased compared to the vector control. In conclusion, inhibition of IGF-IR resulted in suppression of adhesion, invasion, and metastasis, providing a mechanistic rationale for targeting IGF-IR in the treatment of metastatic breast cancer.

Antineoplastic Agents, Phytogenic↗

PubMed1998

Single gene complementation of the hPMS2 defect in HEC-1-A endometrial carcinoma cells.

Results from the analysis of human tumor cell lines with mutations in DNA mismatch repair genes have contributed to the understanding of the functions of these gene products in DNA mismatch repair, microsatellite instability, cell cycle checkpoint control, transcription-coupled nucleotide excision repair, and resistance to cytotoxic agents. However, complementation of human DNA mismatch repair defects by introduction of a single cloned gene or cDNA, which would serve to directly prove or disprove their involvement in these processes, has not been accomplished. Here, we introduce a wild-type copy of the hPMS2 cDNA by stable transfection into the PMS2 mutant HEC-1-A cell line. HEC-1-A cells expressing wild-type hPMS2 exhibit increased microsatellite stability, have a reduced mutation rate at the endogenous hypoxanthine phosphoribosyltransferase locus and extracts from these cells are able to perform strand-specific mismatch repair. These results demonstrate that the hPMS2 gene is integral to the maintenance of genome stability.

Adenosine Triphosphatases↗

PubMed1998

Complementation of mismatch repair gene defects by chromosome transfer.

The study of the multiple functions of mismatch repair genes in humans is being facilitated by the use of human tumor cell lines carrying defined MMR gene mutations. Such cell lines have elevated spontaneous mutation rates and may accumulate mutations in other genes, some of which could be causally related to the phenotypes of these cells. One approach to establish a cause-effect relationship between a MMR gene defect and a phenotype is to determine if that phenotype is reversed when a normal chromosome carrying a wild-type MMR gene is introduced by microcell fusion. This approach has the advantage of presenting the gene in its natural chromosomal environment with normal regulatory controls and at a reasonable dosage. The approach also limits candidate genes to only those encoded by the introduced chromosome and not elsewhere in the genome. Here we review studies demonstrating that hMSH2, hMSH3, hMSH6 and hMLH1 gene defects can each be complemented by transferring human chromosome 2, 5, 2 or 3, respectively. These transfers restore MMR activity, sensitivity to killing by MNNG, stability to microsatellite sequences and low spontaneous HPRT gene mutation rates.

Cell Fusion↗