Biomedical subjects

J C Barrett

Publications and source records attributed to J C Barrett.

At least 199 records · Page 11Linked to original sources

PubMed1995

Aflatoxin B1-induced immortalization of cultured skin fibroblasts from a patient with Li-Fraumeni syndrome.

To examine the mechanisms of immortalization in human cells, normal human diploid fibroblasts (WHE-7) and skin fibroblasts from a patient with Li-Fraumeni syndrome (MDAH 087) and a mutant p53 allele were treated with aflatoxin B1 (AFB1). Exogenous metabolic activation of AFB1 with rat liver post-mitochondrial supernatant (PMS) was used and the optimal treatment conditions needed were determined by the inducibility of unscheduled DNA synthesis. The same degree of cytotoxicity was observed with MDAH 087 cells and normal WHE-7 cells treated with AFB1 at 0.1, 0.3 or 1 microgram/ml for 2 h with a 2% PMS mixture. All WHE-7 cell cultures (AFB1-treated and controls) failed to escape from senescence, whereas three out of nine AFB1-treated cultures of MDAH 087 cells escaped senescence. MDAH 087 cells treated with 0.1 microgram/ml of AFB1 two or three times initially decreased in growth approximately 40 days [10 population doublings (PD)] after the first treatment. However, the cells recovered with faster growth rates after approximately 100 additional days and grew continuously. Both cultures were immortal, defined as continuous growth for over 300 PD. Cells treated once with 0.3 microgram/ml of AFB1 also escaped senescence, although they had about a 230 day time lag before restoration of cell growth. The three AFB1-treated cell lines exhibited altered morphologies, chromosome aberrations (numerical and structural aberrations) and loss of the wild-type p53 allele. Although immortal, the cells were non-tumorigenic in nude mice. Spontaneous immortalization of untreated MDAH 087 was not observed in this study. The results indicate that AFB1 treatment of cells from a Li-Fraumeni patient, but not cells from normal individuals, can induce immortalization. This model may be useful for studying mechanisms of chemically induced immortalization.

Adult↗

PubMed1995

Chemically induced mammary gland cancer in the National Toxicology Program's carcinogenesis bioassay.

Incidences of breast cancer change in populations as people migrate from one area of the world to another, suggesting that environmental factors contribute to this disease. There is a continuing effort to identify these environmental factors and the role that exposures to specific chemicals play in this disease. Results from experimental studies show that chemicals identified to cause mammary gland cancer in rodents are frequently mutagenic chemicals, suggesting that genetic damage is an important mechanism for the induction of this cancer. Prevalent classes of chemicals that were identified to cause mammary gland cancer in rodents in studies by the National Toxicology Program include halogenated hydrocarbons, aromatic amino/nitro compounds and epoxide-forming chemicals. Results from these experimental studies will help to elucidate mechanisms and possible causes of breast cancer in humans.

Animals↗

PubMed1995

Effects of testosterone, testosterone propionate, 17 beta-trenbolone and progesterone on cell transformation and mutagenesis in Syrian hamster embryo cells.

Testosterone, testosterone propionate, 17 beta-trenbolone and progesterone, which represent the main endogenous and synthetic androgens and a progestin, were evaluated for possible cell transformation and genetic effects in Syrian hamster embryo (SHE) cells. Cell growth was reduced by treatment with the steroids at 10-30 micrograms/ml in a dose-related manner. Testosterone and testosterone propionate were less toxic than the other two steroids. Testosterone, testosterone propionate and progesterone induced morphological transformation of SHE cells with similar transformation frequencies. The most potent effects were observed with testosterone propionate, which induced cell transformation at 1-30 micrograms/ml in a dose-related manner. Testosterone and progesterone transformed cells only at the highest dose (30 micrograms/ml). 17 beta-Trenbolone did not induce a statistically significant level of cell transformations at any dose tested (up to 30 micrograms/ml). The transformation frequencies induced by testosterone, testosterone propionate and progesterone were less than one-half that induced by benzo[a]pyrene at 1 microgram/ml. None of these steroids induced significant increases in frequencies of chromosome aberrations or aneuploidy. Gene mutations were not observed for testosterone at the HPRT or Na+/K+ ATPase locus. Because these steroids are also associated with carcinogenic activity in vivo, these in vitro findings provide a model and new insights into the study of the mechanisms of androgen- and progestin-induced cell transformation.

Animals↗

PubMed1995

Restoration of the cellular senescence program and repression of telomerase by human chromosome 3.

Telomeres, at the end of chromosomes, shorten with each cell division, resulting in cellular senescence. Tumor cells, unlike normal somatic cells, express a telomerase that maintains the telomere length. Deletion of a gene(s) on chromosome 3 is common in human renal cell carcinoma (RCC) and reintroduction of a normal chromosome 3 into an RCC immortal cell line restored the program of cellular senescence. The loss of indefinite growth potential was associated with the loss of telomerase activity and shortening of telomeres in the RCC cells with a normal chromosome 3. However, microcell hybrids that escaped from senescence and microcell hybrids with an introduced chromosome 7 or 11 maintained telomere lengths and telomerase activity similar to those of the parental RCC23. Thus, restoration of the cellular senescence program by chromosome 3 is associated with repression of telomerase function in RCC cells.

Base Sequence↗

PubMed1995

Homozygous deletions at chromosome 9p21 and mutation analysis of p16 and p15 in microdissected primary non-small cell lung cancers.

Loss of heterozygosity on chromosome 9p has been detected in many primary human tumors and cell lines, suggesting that this chromosomal arm harbors one or more tumor suppressor genes. The recently cloned p16 and p15 genes, mapped to 9p21, are likely candidates for such tumor suppressors. To map the deletion at chromosome 9p21 in non-small cell lung tumors, we analyzed DNA from 25 tumors and matching normal DNAs at six microsatellite markers that flank the region occupied by the p16 and p15 genes. Loss of heterozygosity of at least one microsatellite marker on chromosome 9p21 was detected in 13 (52%) of 25 tumors, including one tumor that exhibited homozygous deletion of both human IFNalpha and D9S171. Six tumors analyzed by a comparative multiplex PCR technique showed homozygous deletions of the sequence tag site marker c5.1 (within p16). Screening for mutations in p16 and p15 revealed one tumor with a non-sense mutation in exon 2 of p16, but no mutations were detected in p15 in any of the tumors. Thus, in these analyses approximately one-half of the non-small cell lung tumors had loss of heterozygosity at chromosome 9p21, and of these tumors, one-half had homozygous deletions of the region that includes p16. This appears to confirm the importance of a locus in this region critical to growth control in lung. The apparent lack of other mutations in p16 and p15 in the tumors with loss of heterozygosity leaves open the possibility of an unidentified gene in this region that may function as a tumor suppressor.

Adenocarcinoma↗

PubMed1994

Evidence for multiple pathways to cellular senescence.

Normal cells in culture generally senesce whereas tumor-derived cells are often, but not without exception, immortal and grow indefinitely. For cells to escape the senescence program, normal genes must be lost or inactivated as shown by somatic cell genetic studies. For example, the introduction of specific chromosomes by microcell-mediated chromosome transfer has been shown to induce senescence of human and rodent tumor cell lines, and the mapping of over ten senescence genes has been achieved by this method. In this study, we observed that two different normal chromosomes induce senescence in the same human endometrial carcinoma cell line, which suggests that multiple pathways to senescence are inactivated in this cell line. This hypothesis has implications for the mechanisms of cellular senescence and its role in carcinogenesis. Furthermore, this hypothesis can explain why not all tumor-derived cells are immortal.

Animals↗

PubMed1994

Differential suppression of mammary and prostate cancer metastasis by human chromosomes 17 and 11.

Metastasis suppressor activities have previously been mapped to human chromosomes 17 and 11. Decreased expression of the metastasis suppressor gene NM23, which is located on chromosome 17, has been correlated with increased metastatic potential in mammary cancers. A region on human chromosome 11, from 11p11.2-p13, has been shown to suppress metastasis in rat prostatic carcinoma cells. In both cases the metastasis suppressor activity had no effect on tumorigenicity or tumor growth rate, demonstrating that the encoded activities are distinct from effects of tumor suppression. To determine whether these human chromosomes encode general or tissue-specific metastasis suppressor activities, a truncated human chromosome 17 (i.e., pter-q23) and a full-length human chromosome 11 were separately transferred into highly metastatic rat mammary and prostate cancer cell lines and tested for their ability to suppress spontaneous metastasis in vivo. These studies demonstrated that when the pter-q23 region of human chromosome 17 is retained by the microcell hybrids, the metastatic ability of both mammary and prostatic cancer cells is suppressed. In contrast, when the pter-q14 region of human chromosome 11 is retained, only the metastatic ability of prostatic cancer cells is suppressed. Additional studies demonstrated that the metastasis suppressor activity encoded by the chromosome 17 pter-q23 region is p53-independent and not due to enhanced expression of NM23 protein.

Animals↗

PubMed1994

Allelotype of endometrial carcinoma.

An allelotype analysis of endometrial carcinoma was undertaken to identify chromosomal loci that are relevant to this tumor type. A total of 70 highly polymorphic microsatellite markers, distributed among all nonacrocentric chromosome arms, were examined for evidence of loss of heterozygosity or allelic imbalance in DNA samples from matched normal and tumor tissues. An average of 21 informative tumor cases were obtained for each marker. Allelic deletions or imbalance were observed on 31 of 41 chromosome arms with no marker showing an allelic loss ratio of greater than 33%. Those chromosome arms most frequently involved were 3p, 8p, 9p, 14q, 16q and 18q. There was a strong correlation between loss of heterozygosity on chromosome 14q and death from disease. These data indicate that the molecular genetic character of endometrial carcinoma is complex and that a relatively large number of different chromosomal loci are likely to play a role in the etiology and progression of this tumor type.

Chromosomes, Human, Pair 14↗

PubMed1994

Regulation of apoptosis by low serum in cells of different stages of neoplastic progression: enhanced susceptibility after loss of a senescence gene and decreased susceptibility after loss of a tumor suppressor gene.

A cell culture model system has been used to study the susceptibility of cells to apoptotic cell death during different stages of neoplastic progression. This system consists of normal diploid Syrian hamster embryo (SHE) cells, two preneoplastic cell lines [tumor suppressor stage I (sup +I) and non-tumor suppressor stage II (sup -II)], and hamster tumor cell lines. Stage I preneoplastic cells are nontumorigenic immortal clones that suppress tumorigenicity when hybridized to tumor cells, whereas stage II cells have lost the ability to suppress tumorigenicity in cell hybrids. We refer to these two types of preneoplastic cells as sup +I and sup -II, respectively. Neoplastic progression is generally associated with cellular alterations in growth factor responsiveness. Therefore, to study the regulation of apoptosis in the system described above, cells were cultured in low serum (0.2%) as a means of withdrawing growth factors. In low serum, normal SHE cells were quiescent (labeling index of 0.2%), with little cell death. The sup +I cells showed a relatively low labeling index (1.6%) but, in contrast to the normal cells, died at a high rate (55% cell loss after 48 h) by apoptosis, as evidenced by morphology, DNA fragmentation, and in situ end-labeling of fragmented DNA. The apoptotic cells did not go through a replicative cycle while in low serum, implying that apoptosis was initiated in the G0/G1 phase of the cell cycle. The sup -II cell line showed a high labeling index (40%) after 48 h, but cell growth was balanced by cell death that occurred at approximately the same rate. The cells died, however, predominantly by necrosis. The tumor cell lines continued to proliferate in low serum, with high labeling indices (ranging from 27% to 43%) and a low level of apoptotic or necrotic cell death. To determine the relative ability of these cells to survive in vivo, normal SHE cells, sup +I cells, and sup -II cells were injected s.c. into nude mice. At 5 or 21 days after injection, the normal SHE cells were readily retrieved from the mice and grew well in culture. In contrast, few sup +I cells were retrieved 5 days after injection and no viable cells were retrieved after 21 days. Sup -II cells were not retrieved at either the 5-day or 21-day harvest, and histological examinations of the sites of injection showed the presence of macrophages, eosinophils, and neutrophils, indicating an inflammatory response associated with necrotic cell death.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗

PubMed1994

Differential proliferative responses of Syrian hamster embryo fibroblasts to paraquat-generated superoxide radicals depending on tumor suppressor gene function.

Oxygen radicals have been widely implicated in neoplastic transformation; however, little is known regarding their mode of action. In an attempt to delineate potential mechanisms of action, an analysis of superoxide effects on cell growth was studied in normal and two nontumorigenic, immortal cell lines derived from normal Syrian hamster embryo (SHE) fibroblasts. The two immortal cell lines differed in their ability to suppress tumorigenicity of tumor cells in cell hybrids. One cell line suppressed tumorigenicity (sup+), while a second clone was unable to suppress tumorigenicity (sup-). Paraquat was used to generate superoxide through its capacity to be reduced by NAD(P)H and to generate superoxide radicals. The growth response of the various cell types was measured by colony-forming ability as well as by tritiated thymidine incorporation using autoradiography. At low paraquat concentrations (25 microM), primary SHE cells and two sup+ clones showed up to a 40% enhancement in colony formation, while two sup- clones showed no increase. Toxicity was observed at high doses, starting at approximately 100 microM paraquat. Since oxygen radicals are also mutagenic, primary SHE cells were examined for chromosomal aberrations. Chromatid gaps and breaks were induced at all concentrations of paraquat used. Thus, superoxide not only causes cellular toxicity at high doses but at low doses enhances cell growth of certain cells (primary SHE cells and sup+ cells) but not others (sup- cells). Therefore, differing responses of cells at different stages of neoplastic progression must be considered in understanding oxygen radical effects in growth control and carcinogenesis.

Animals↗

PubMed1994

Disruption of G0-G1 arrest in quiescent and senescent cells treated with phosphatase inhibitors.

The majority of signal transduction studies have focused on events induced by mitogen stimulation. However, little is known about the negative control signals that cause or maintain growth arrest and must be overcome for mitogenesis to occur. We investigated the possible role of protein phosphatases in this negative regulatory process. Treatment of quiescent hamster and human fibroblasts with low doses of the phosphatase inhibitors sodium o-vanadate or okadaic acid allowed 30-40% of cells to progress from G0-G1 arrest to S phase. This was accompanied by phosphorylation of the retinoblastoma and MAP-kinase proteins, as well as induction of the cdc2 protein. Furthermore, we observed that protein phosphatase inhibitor treatment could override the block to DNA synthesis in senescent cells, which are normally nonresponsive to mitogens. These data suggest that protein phosphatases may play a role in the negative regulation of cell growth and maintenance of growth arrest.

Animals↗

PubMed1994

Mutation analysis of the THRA1 gene in breast cancer: deletion/fusion of the gene to a novel sequence on 17q in the BT474 cell line.

We have previously described a common region of deletion and allele loss on chromosome 17q in sporadic breast cancers that is likely to contain a tumor suppressor gene. The region, mapped to 17q12-q21, was bordered by D17S250 and D17S579 on the centromeric and telomeric sides, respectively. This deletion region overlaps the BRCA1 locus, which predisposes to familial breast and ovarian cancer. The most frequent loss of heterozygosity was observed at the thyroid hormone receptor alpha (THRA1) locus. Southern analysis revealed a rearrangement of THRA1 in the BT474 breast cancer cell line. This rearrangement represented a deletion of exons 8-10 of one THRA1 allele that was also coamplified with ERBB2. Northern blots showed two mutant transcripts in BT474 cells. Analysis of the mutant transcripts revealed fusion of the THRA1 exon 7 by splicing to a novel sequence designated BTR for "BT474 transcribed rearrangement." BTR was found to be highly conserved and mapped to 17q. The deletion in BT474 cells spans the entire BRCA1 region. To search for additional mutations in the THRA1 gene, all nine protein-encoding exons of THRA1 were examined for point mutations via single strand conformation analysis in a series of primary breast tumors, breast cancer cell lines, and lymphoblastoid cell lines derived from the youngest affected members of several German breast cancer families. No point mutations were detected, including the unrearranged THRA1 allele in BT474. We have thus excluded THRA1 as a commonly mutated sporadic breast cancer tumor suppressor gene and as the BRCA1 gene.

Amino Acid Sequence↗

PubMed1994

Increased actin cable organization after single chromosome introduction: association with suppression of in vitro cell growth rather than tumorigenic suppression.

We previously showed that introduction of a single human chromosome 1, 6, or 9 derived from normal fibroblasts into HHUA endometrial carcinoma cells resulted in suppression of tumorigenicity. The tumorigenic suppression was accompanied by remarkable morphological changes in the microcell hybrids containing an extra copy of chromosome 1. The study presented here was undertaken to search for target cytoskeletal components affected by chromosome 1 transfer into endometrial carcinoma cells. We found that the microcell hybrids containing an extra copy of chromosome 1 were characterized by intracellular actin bundle formation and an excessive accumulation of actin and vinculin. The latter was a result of increased stabilization of the proteins. Additionally, chromosome 3 introduction into RCC23 human renal carcinoma cells resulted in prolongation of cell division and in senescence of a significant proportion of the microcell hybrids. In these microcell hybrids, the intracellular actin network was also reorganized, but the amounts of actin and vinculin protein were not increased. These findings suggest that the increased actin organization, which appeared not to cause tumorigenic suppression in the microcell hybrids, is associated with complementation of tumor suppressor genes and senescence by multiple mechanisms.

Actin Cytoskeleton↗

PubMed1994

Evidence for a p53-independent pathway for upregulation of SDI1/CIP1/WAF1/p21 RNA in human cells.

SDI1 is an inhibitor of DNA synthesis that we isolated by expression screening cDNAs prepared from senescent, terminally nondividing human cells. Other groups then cloned this gene as a cyclin-dependent kinase (cdk)-interacting protein (CIP1, p21) that inhibits cdks; the gene was also isolated by screening for genes transactivated by p53 (WAF1). p53 levels are low in senescent and quiescent contact-inhibited or serum-deprived normal human cells, which we have found express high levels of SDI1 mRNA. This indicates that alternate pathways for upregulation of message level of this gene may exist. We therefore proceeded with the study presented here, treating human cells with a variety of growth-arrest-inducing agents, including some that damaged DNA, and found that RNA levels of SDI1 were increased in all cases that resulted in growth inhibition. More important, with the exception of gamma-radiation, most of these agents were able to elevate SDI1 message levels in cells lacking wild-type p53. At least two distinct kinetic profiles for RNA induction were observed, one that implicated p53 transactivation and occurred early enough to cause arrest, and another that clearly was p53 independent and suggested a role for the SDI1 gene product in the maintenance rather than in the cause of inhibition of DNA synthesis.

Antimetabolites↗

PubMed1994

Cytogenetic analysis of murine cell lines from diethylstilbestrol-induced uterine endometrial adenocarcinomas.

Treatment of female CD-1 mice with the synthetic estrogen diethylstilbestrol (DES) on days 1 through 5 after birth results in a 90% incidence of endometrial adenocarcinomas by 18 months of age Three cell lines were established from DES-induced uterine carcinomas and studied for specific chromosomal changes. Each cell line exhibited numerical decreases in chromosomes 9, 11, 13, and X as common abnormalities. Structural alterations involving chromosomes 3, 6, 11, and 19 occurred nonrandomly among the three cell lines. Every cell line showed a rearrangement in the long arm of chromosome 3 (3q+), a translocation between chromosomes 3 and 19 [t(3;19)], isochromosome of chromosome 11 [i(11)], and a marker chromosome (M2) either as common abnormalities or recurrent abnormalities. t(3;19), i(11), and M2 were observed also in the primary colonies from which the cell lines arose. The changes were not observed in a cell line derived from the uterus of one untreated control mouse, suggesting that these chromosomal alterations may have occurred during DES-induced neoplastic transformation. The chromosomal alterations found in the present study may prove useful in investigating the genetic changes involved in DES carcinogenesis.

Adenocarcinoma↗

PubMed1994

Reserpine-induced cell transformation without detectable genetic effects in Syrian hamster embryo cells in culture.

Reserpine, a naturally occurring rauwolfia alkaloid, used mainly as an antihypertensive drug, was examined for its ability to induce cell transformation and genetic effects in Syrian hamster embryo (SHE) cells in culture. Treatment of SHE cells with 2 micrograms/ml of reserpine had no effect on cell growth, while 4 micrograms/ml of reserpine reduced the growth rate slightly and 8 micrograms/ml resulted in a significant inhibition of cell growth. Reserpine at doses of 4-8 micrograms/ml for 48 h induced a dose-related increase in morphological transformation of the cells. Reserpine-transformed colonies were morphologically indistinguishable from colonies transformed with benzo[a]pyrene (B[a]P) or other chemical carcinogens. Over the dose range that resulted in cell transformation, treatment of SHE cells with reserpine failed to induce any detectable gene mutations at two genetic loci, chromosomal abnormalities including structural and numerical changes, or DNA adduct formation. These findings indicate that reserpine may have carcinogenic potential by unknown mechanisms that do not include direct induction of gene and/or chromosome mutations.

Aneuploidy↗

PubMed1994

Loss of heterozygosity in spontaneous and chemically induced tumors of the B6C3F1 mouse.

The B6C3F1 mouse is used worldwide to gauge the carcinogenic hazard posed by chemicals to humans. An assessment of the ability of this rodent model to predict human neoplasia requires an evaluation of similarities and differences in the genetics of tumor formation between these two species. We examined 142 spontaneous and chemically-induced liver tumors isolated from the B6C3F1 mouse for losses of heterozygosity (LOH) at 78 polymorphic loci and compared these results to genetic changes known to occur in human hepatocellular carcinoma. Approximately a third of the 142 mouse tumors exhibited LOH, suggesting that tumor suppressor gene inactivation may be involved in the formation of mouse liver tumors. Most of the LOH observed was restricted to seven chromosome sites and most of the tumors that underwent LOH lost alleles from only one of those seven sites. The relatively few losses seen in these mouse tumors distinguished them from clinical stage human tumors in that, in the mouse tumors, interstitial deletions appeared more frequently than losses of whole chromosomes. Only four mouse tumors lost a whole chromosome. LOH occurred at loci of the mouse genome syntenic to areas of the human genome known to harbor the Wilms', retinoblastoma, APC, MCC and DCC tumor suppressor genes; these genes have never been associated with hepatocellular carcinomas. Losses observed on chromosomes 5 and 8 (syntenic to human chromosomes 4 and 16) suggest tumor suppressor genes that are common to hepatocellular carcinomas from both species, while losses on chromosome 9 suggest involvement of a previously unidentified tumor suppressor gene.

Animals↗

PubMed1994

Utilization of a fos-lacZ plasmid to investigate the activation of c-fos during cellular senescence and okadaic acid-induced apoptosis.

C-fos is an immediate-early gene that is induced by external stimuli and is possibly involved in initiation of DNA synthesis by such stimuli. In these studies, we used the murine c-fos promoter coupled to a lacZ reporter gene to study fos induction in senescent and quiescent cells. In transfected, quiescent, immortal Syrian hamster embryo (SHE) cells (10W), serum stimulation induced the expression of the fos construct to the same extent that DNA synthesis was stimulated. In contrast, in transfected normal cells that have a finite life span, we observed that the cells failed to display upregulation of fos-lacZ in response to serum in individual cells as they senesced. High doses of the phosphatase inhibitor okadaic acid (160-1000 nM) also induced fos-lacZ expression in quiescent immortal cells; however, induction of DNA synthesis and expression of fos-lacZ were not coordinately induced as a function of okadaic acid concentration. Low concentrations of okadaic acid (0.16 nM) induced DNA synthesis but not fos-lacZ expression, indicating that induction of DNA synthesis by phosphatase inhibitors may bypass, at least quantitatively, the requirement for c-fos induction. At the levels of okadaic acid that induced fos-lacZ expression, cell death, rather than DNA synthesis, was observed. The cells died by apoptosis, thereby implicating a signaling pathway that includes c-fos induction in this process.

Animals↗