Mechanical load stimulates expression of novel genes in vivo and in vitro in avian flexor tendon cells.

OBJECTIVE: Our experiments were designed to test the hypothesis that tendon cells might respond differently to applied strain in vitro than in vivo. DESIGN: We tested cells in whole tendons from exercised chickens and from isolated surface (TSC) and internal tendon (TIF) in vitro that were subjected to mechanical strain. We hypothesized that tendon cells differentially express genes in response to mechanical loading in vivo and in vitro. METHODS: We utilized an in-vivo exercise model in which chickens were run on a treadmill in an acute loading regime for 1 h 45 min with the balance of time at rest to 6 h total time. Gene expression was analyzed by a differential display technique. In addition, isolated avian flexor digitorum profundus TSC and TIF cells were subjected to cyclic stretching at 1 Hz, 5% average elongation for 6 h, +/- PDGF-BB, IGF-I, TGF-beta 1, PTH, estrogen, PGE2, or no drug and/or no load. mRNA was then collected and samples were subjected to differential display analysis. CONCLUSIONS: Load with or without growth factor and hormone treatments induced expression of novel genes as well as some known genes that were novel to tendon cells. We conclude that the study of gene expression in mechanically loaded cells in vivo and in vitro will lead to the discovery of novel and important marker proteins that may yield clues to positive and negative cell strain responses that are protective under one set of conditions and destructive under another.

Mesotheliomas induced in rats by the fibrous mineral erionite are independent from p53 alterations.

The development of human malignant mesothelioma (MM) is strongly associated with occupational or environmental exposure to certain natural mineral fibers, although the genetic mechanisms underlying this malignancy remain unclear. Although the p53 gene is frequently mutated in various tumors, human asbestos-associated MMs appear to develop independently from p53 alterations. The high mesotheliomagenic potency of natural fibrous mineral erionite is well established in humans and rodents, but no data regarding genetic alterations in erionite-associated tumors are currently available. Previous speculations that the oncogenic mechanisms underlying asbestos and erionite carcinogenesis may differ led us to examine whether the p53 gene is targeted in erionite carcinogenesis. Fifteen erionite-induced rat MMs as well as six cell lines derived from asbestos-induced and spontaneous rat MM were analyzed for p53 mutations by direct DNA sequencing and immunohistochemical analysis. Both approaches did not reveal p53 alterations in rat MM samples used in the study indicating that, similar to asbestos carcinogenesis, erionite carcinogenesis does not target the p53 tumor suppressor gene.

Differential display identified changes in mRNA levels in regenerating livers from chloroform-treated mice.

The nongenotoxic-cytotoxic carcinogen chloroform induces liver necrosis, regenerative cell proliferation, and, eventually, liver tumors in female B6C3F1 mice when administered by gavage at doses of 238 or 477 mg/kg/d. Administration of 1800 ppm of chloroform in the drinking water results in similar daily doses but does not produce liver toxicity or cancer. The differential-display technique was used to compare the expression of a subset of mRNAs in normal (control) and regenerating liver after chloroform-induced toxicity to define the proportion of genes whose expression changes under hepatotoxic conditions and to identify the genes that might play a role in regeneration and perhaps cancer. RNA was purified from the livers of female B6C3F1 mice after 4 d or 3 wk of gavage treatment with 3, 238, or 477 mg/kg/d of chloroform or treatment with 1800 ppm chloroform in drinking water. There was a remarkably high degree of consistency of gene expression among the animals and across dose and treatment groups as visualized by the differential-display technique. Of the 387 bands observed, only four (about 1%) changed expression in regenerating liver. The genes were assigned locus names by GenBank after sequence submission. The genes with increased mRNA levels as confirmed by northern blot analysis were MUSTIS21, a mouse primary response gene induced by growth factors and tumor promoters; MUSMRNAH, a gene highly homologous to a human gene isolated from a prostate carcinoma cell line; and MUSFRA, a novel gene. The novel gene MUSFRB exhibited decreased mRNA levels. No change in expression was seen among control mice given the nontoxic regimens of 3 mg/kg/d chloroform or 1800 ppm chloroform in drinking water, indicating that changes in expression were associated with toxicity and regeneration rather than chloroform per se. These genes and others that may be identified by expanding this approach may play a role in regeneration and perhaps in the process of chloroform-induced carcinogenesis in rodent liver.

p53 status in spontaneous and dimethylnitrosamine-induced renal cell tumors from rats.

Rats carrying the Eker tumor-susceptibility mutation (Eker rats) are predisposed to developing renal cell carcinoma. Rats heterozygous for the Eker mutation develop spontaneous multiple bilateral renal cell tumors by the age of 1 yr. In a previous study, Eker-mutation carrier and noncarrier rats were exposed to the renal carcinogen dimethylnitrosamine (DMN), and male rats carrying the Eker mutation exhibited a 70-fold increase in the induction of renal adenomas and carcinomas when compared with noncarrier rats. In this study, spontaneous and DMN-induced rat renal cell tumors (adenomas and carcinomas) were analyzed for mutations of the p53 gene by direct sequencing of cDNA polymerase chain reaction products. There were no mutations in p53 cDNA derived from renal tumors from six untreated rats. Mutations were found in one of 15 of the DMN-induced tumors: a transition at codon 140, CCT-->CTT, in a renal adenoma. Additionally, seven cell lines derived from spontaneous renal cell tumors did not contain mutations in p53. The low frequency of p53 mutations (one of 21 renal cell tumors and none of seven cell lines derived from renal cell tumors) indicates that the development of both spontaneous and carcinogen-induced renal tumors involved a non-p53-dependent pathway. As p53 is infrequently mutated in human renal cell carcinomas and in rat renal mesenchymal tumors, it is likely that a tumor suppressor gene or genes other than p53 are involved in the development of renal cancer.

Increased susceptibility to in vitro transformation of cells carrying the Eker tumor susceptibility mutation.

Rats carrying the Eker tumor susceptibility mutation are genetically predisposed to renal cell carcinoma. Rats heterozygous for the Eker mutation (Eker carriers) develop multiple bilateral renal cell carcinomas by the age of 1 year. Using an in vitro rat kidney epithelial (RKE) transformation assay developed in our laboratory, proximal tubule cells derived from known Eker rat carriers (+/ek) and non-carriers (+/+) were exposed to the carcinogen N-methyl-N'-nitro-N-nitrosoguanidine (MNNG), to determine if cells derived from Eker carriers were more susceptible to in vitro transformation than cells derived from non-carrier animals. The percent transformation frequency following MNNG treatment was 7.5-fold higher in cells derived from carrier animals when compared to cells from non-carrier animals. This increased susceptibility to transformation due to inheritance of the Eker mutation is consistent with a predisposition resulting from inactivation of a tumor suppressor gene. The increased susceptibility of kidney epithelial cells carrying the Eker mutation may prove useful in the further development of the RKE transformation assay as a sensitive tool to identify potential renal carcinogens. In addition, because transformation frequency in the RKE assay measures a very early step in multistage transformation, these results also suggest that alterations (by Loss of Heterozygosity or mutation) at the Eker tumor susceptibility locus are an early event in the development of renal tumors in the rat.

The role of O6-alkylguanine in cell killing and mutagenesis in Chinese hamster ovary cells.

Chinese hamster ovary cells with no detectable (less than 200 molecules/cell) O6-methylguanine-DNA methyltransferase (EC 2.1.1.63) were transfected with human cell DNA and pSV2neo plasmid by electroporation. Two stable transformant clones, GC-1 and GC-2, containing 4 X 10(4) and 4-6 X 10(3) methyltransferase molecules/cell respectively were isolated by successive screening in the presence of G418 and 2-chloroethyl-N-nitrosourea (CNU). Only three or four copies of pSV2neo DNA and no repetitive human DNA sequence were detected in these isolates. Secondary transfection of parent cells with GC-1 DNA yielded several clones containing 2-10 X 10(3) methyltransferase molecules/cell. The rate of removal of O6-methylguanine in GC-1, GC-2 and parent cells in vivo reflected their methyltransferase levels, while the N-methylpurines were removed at similar rates in all three cell lines. The differential sensitivity of these cells to several alkylating agents, namely CNU, N-methyl-N-nitrosourea, N-methyl-N'-nitro-N-nitrosoguanidine and methyl-methane sulfonate (MMS), known to yield different proportions of O6-alkylguanine among the alkyl adducts in DNA, varied widely. The largest and smallest differences in toxic response were observed with CNU and MMS respectively. These cell lines showed no difference in sensitivity to the DNA cross-linking agent psoralen. These data strongly suggest that alkylating agents produce two classes of lethal lesions, one of which is O6-alkylguanine. Induction of mutations at the hypoxanthine-phosphoribosyltransferase locus in these cells lines suggests that, regardless of its relative yield, O6-methylguanine is the major mutagenic lesion for all alkylating agents.