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C L Parfett

Publications and source records attributed to C L Parfett.

At least 19 recordsLinked to original sources
PubMed2000

Promotion of morphological transformation by Di-n-butyltin dichloride in C3H/10T1/2 cells: prediction by prior expression of tumour promoter-responsive genes.

Previous studies in our laboratory have shown that chemical treatments may induce increases in proliferin gene family mRNA accumulation in cultured murine embryonic cells. Proliferin inductions are highly correlated with subsequent promotional outcomes during two-stage focus-formation assays in C3H/10T1/2 cell cultures. In work reported here, the strong affiliation between these two responses was further validated after treating cells with di-n-butyltin dichloride which is a polyvinyl chloride (PVC) plastic additive that often contaminates food and water. Increased proliferin expression and promotion of morphological transformation occurred at similar concentrations. Promotion of transformation was detected at di-n-butyltin dichloride concentrations of 80 nM (24 ng/ml) and above, if added to initiated cultures before confluent monolayers had formed. Proliferin induction and morphological transformation were both reduced in confluent cultures treated with di-n-butyltin dichloride, as compared to subconfluent cultures. Proliferin expression measured in near-confluent cultures was induced up to 10-fold during the 36-hr period following di-n-butyltin dichloride exposure and was accompanied by increased accumulation of transcripts from many genes regulated by oxidative stresses, growth-inducing agents, and/or other promoting agents (asbestos, superoxide radicals ). Di-n-butyltin dichloride-induced mRNA species included members of the fos and jun proto-oncogene families, c-myc, egr1, ribonucleotide reductase (R2 subunit), odc, macrophage chemotactic protein/je, hsp70, metallothionine IIA, c-sod and mn-sod. The observed patterns of RNA accumulation suggested that a small subset of mRNA species, including proliferin, exhibit regulatory behaviour as a response to dissimilar agents or conditions that promote focus-formation in C3H/10T1/2 cultures. Plausible predictions of promotional effects in two-stage morphological transformation assays can be made from gene-expression responses to test agents.

Animals↗
PubMed1996

Asbestos promotes morphological transformation and elevates expression of a gene family invariably induced by tumor promoters in C3H/10T1/2 cells.

The murine proliferin gene family, which has been shown to respond consistently to tumor promoters and other cellular pro-oxidant agents in C3H/10T1/2 cells, was used to monitor responses after treatment of these cell cultures with toxic, pro-oxidant asbestos fibres. Proliferin mRNA levels were increased by amosite, crocidolite or chrysotile asbestos fibres, especially in the presence of fresh serum and at low cell densities. Promotion of morphological transformation was confirmed in two-stage focus formation assays using crocidolite at a fibre density that induced proliferin expression. Asbestos-induced gene expression was inhibited by millimolar levels of N-acetylcysteine (NAC), supporting a linkage between: (i) induced oxidant stress that was sufficient to promote morphological transformation; (ii) induction of proliferin expression. Other anti-oxidant compounds (dithiothreitol and pyrrolidine dithiocarbamate) or enzymes (superoxide dismutase and catalase) did not inhibit induced expression. Non-fibrous powders (titanium dioxide, quartz or silica gel) were also effective inducers of proliferin mRNA accumulation. Latex beads and activated charcoal were effective at higher particle densities, implying that ubiquitous particle-induced surface membrane effects can lead to an NAC-reversible step necessary for proliferin induction. The results showed that asbestos resembled all other promoters of morphological transformation in C3H/10T1/2 cells in that an antioxidant-sensitive induction of the proliferin gene family occurred following treatment.

Animals↗
PubMed1995

Oxidative stress-regulated gene expression and promotion of morphological transformation induced in C3H/10T1/2 cells by ammonium metavanadate.

Promoters of C3H/10T1/2 cell morphological transformation that elevate intracellular oxidant levels can be distinguished by a spectrum of induced gene expression, which includes the oxidant-responsive murine proliferin gene family. Proliferin transcripts were induced 40- to 100-fold by 20 microM ammonium metavanadate, 20-fold by 5 microM vanadium pentoxide but only three-fold by vanadium oxide sulfate. Consistent with its response to other oxidant chemicals, induction of proliferin by ammonium metavanadate was inhibited almost completely by the antioxidant N-acetylcysteine (8 mM). Ammonium metavanadate (5 microM), added as promoter in two-stage morphological transformation assays, amplified yields of Type II and Type III foci in monolayers of 20-methylcholanthrene-initiated C3H/10T1/2 cells. Ammonium metavanadate also induced formation of Type II foci in single-step transformation assays. The results suggest that pentavalent vanadium compounds could promote morphological transformation in C3H/10T1/2 cells by creating a cellular state of oxidative stress, sufficient to induce elevated expression of the proliferin gene family.

Animals↗
PubMed1993

Tri-n-butyltin chloride promotes morphological transformation and induces proliferin expression in C3H10T1/2 cells.

Transcripts from the murine gene family proliferin, which are increased by a wide assortment of chemical promoters of C3H10T1/2 cell morphological transformation, were shown to be induced by tri-n-butyltin chloride at concentrations above 50 nM. Two-stage transformation assays, with 3-methylcholanthrene as inducer and tri-n-butyltin chloride as promoter, were performed to determine if promotion of morphological transformation and proliferin induction were properties shared by this compound. Tri-n-butyltin chloride synergistically enhanced focus formation at concentrations ranging from 20 to 75 nM. Di-n-butyltin dichloride, n-butyltin trichloride and tin (II) chloride, but not tin (IV) chloride, were also effective inducers of proliferin. Changes in patterns of TPA-inducible, secreted proteins, including those likely to be proliferin, were detected following organotin treatment of confluent monolayers. Tri-n-butyltin chloride resembles other agents active as promoters in C3H10T1/2 two-stage transformation assays by possessing an ability to induce proliferin expression.

Animals↗
PubMed1992

Induction of proliferin gene expression by diverse chemical agents that promote morphological transformation in C3H/10T1/2 cultures.

Anthralin, iodoacetic acid, BHT, Tween 60 and TPA induced cytoplasmic accumulation of transcripts of the proliferin gene family at or near effective concentrations for promotion of morphological transformation of C3H/10T1/2 cells. Conversely, non-promoters (4-alpha-phorbol, formic acid and methanol) were ineffective inducers. Compounds likely to promote transformation (nickel sulphate, benzyl peroxide and t-butyl hydroperoxide) were also effective and apparently selective, proliferin inducers in C3H/10T1/2 and primary murine fibroblasts. Proliferin induction may be a useful molecular marker to predict chemically-induced promotion of morphological transformation in C3H/10T1/2 cells.

Animals↗
PubMed1991

Spontaneous immortalization of mouse embryo cells: strain differences and changes in gene expression with particular reference to retroviral gag-pol genes.

We have studied the kinetics with which cultures of primary mouse embryo cells pass through the crisis period, escape their terminal differentiation (cellular senescence), and give rise to an immortal cell line. The process is strain-dependent, with cells from the outbred Swiss CD-1 mouse being considerably more adept at forming an immortal 3T3 line than cells from the inbred SWR line; Balb/c cells appeared intermediate in their behavior. The continued presence of the tumor promoter 12-O-tetradecanoylphorbol-13-acetate or the poly(ADPribose)polymerase inhibitor 3-aminobenzamide affected the kinetics but did not seem to alter the outcome. Changes in expression of various genes, including those encoding mitogen-regulated protein (proliferin), endogenous gag-pol retrovirus sequences, insulin-like growth factor II, and a variety of protooncogenes, were monitored during the process of immortalization, and although certain changes were reproducibly characteristic of cells from a given mouse strain passed according to a specific regimen, none of the observed changes were reproducibly characteristic under all conditions of immortalization. In particular, our data indicate the absence of a strict correlation between cellular immortalization and the activation of endogenous gag-pol expression. We conclude from our observations that the establishment of permanent lines from primary mouse embryo cells in serum-containing medium reflects the selection of a variant subpopulation of cells that did not preexist but rather arose in response to the specific culture conditions by a process resembling differentiation. Multiple and complex changes in gene expression occur that are affected by the culture conditions and the strain (genotype) of the mouse.

Animals↗
PubMed1991

Regulation of the expression of mitogen-regulated protein (MRP; proliferin) and cathepsin L in cultured cells and in the murine placenta.

The genes encoding mitogen-regulated protein (MRP; also called proliferin; PLF) and procathepsin L (CL; also called major excreted protein; MEP) are expressed to high levels in the mouse placenta. Although they are both regulated by epidermal growth factor (EGF) and fibroblast growth factor (FGF) in 3T3 cells, expression of these genes is differently regulated with growth state. The expression patterns of MRP and CL as a function of murine development are also different. Basal and growth factor-stimulated levels of MRP expression are much higher in growing than in quiescent 3T3 cells, whereas CL levels are similar. These changes in gene expression in cultured quiescent cells parallel the changes in MRP and CL expression observed in the late-gestational quiescent placenta. These results suggest growth factors may regulate the expression of these genes, but other influences also regulate the expression of MRP and CL in vivo.

3T3 Cells↗
PubMed1990

Enhanced accumulation of mRNA for 78-kilodalton glucose-regulated protein (GRP78) in tissues of nonobese diabetic mice.

Using 78-kildalton glucose-regulated protein cDNA as a probe of Northern blots, we have examined the distribution and inducibility of mRNA encoding the 78-kilodalton glucose-regulated protein in three tissues of nonobese diabetic mice. The gene was constitutively expressed in normal, unstressed cells of liver, brain, and spleen. Developing diabetes correlated with elevated expression in only liver and brain of diabetic mice. This induction of gene expression was associated with the transition from the prediabetic stage to the onset of hyperglycemia and coincided with falling levels of plasma insulin and rising hyperglycemia. The activation of 78-kilodalton glucose-regulated protein gene expression appeared to be transient. We suggest that the temporally differential, tissue-specific expression of this gene in adult nonobese diabetic mice offers an opportunity to study a physiologically relevant regulation of this stress-induced gene.

Animals↗
PubMed1990

Identification of a serum-inducible messenger RNA (5B10) as the mouse homologue of calcyclin: tissue distribution and expression in metastatic, ras-transformed NIH 3T3 cells.

A mouse mRNA, provisionally designated 5B10, has been cloned based on its inducibility by serum in quiescent murine fibroblasts. Here we report the full-length complementary DNA sequence and a partial characterization. There are about five copies of the gene in the mouse genome. Sequence analysis of the 5B10 coding region reveals 94 and 97% amino acid identity to human and rat calcyclin, respectively. Although the coding region has been highly conserved during evolution of the rodent and human genomes, the untranslated flanking sequences differ significantly. A protein of Mr about 8000 was produced by in vitro translation of the mRNA transcribed in vitro from 5B10 complementary DNA in a riboprobe vector. An antiserum raised against a portion of the predicted human calcyclin protein cross-reacted with this mouse protein. 5B10 mRNA was found in greatest amount in organs containing proliferating cells, e.g., epidermis, skin, stomach, uterus of pregnant mouse, placenta, and decidua. Brain, liver, mature thymus, and skeletal muscle had little or no detectable 5B10 mRNA. 5B10 mRNA levels were higher in cells treated with 7,12-dimethylbenzanthracene and 12-O-tetradecanoylphorbol-13-acetate than in their normal counterparts, suggesting a role in tumorigenesis. In addition, high 5B10 mRNA levels were associated with metastatic ability in a series of ras-transformed cells, in proportion to levels of ras p21 expressed by the cells, implicating 5B10 even more deeply in carcinogenesis.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1989

Differential screening of a cDNA library with cDNA probes amplified in a heterologous host: isolation of murine GRP78 (BiP) and other serum-regulated low-abundance mRNAs.

A novel method was used to screen differentially a cDNA library for clones representing serum-regulated mRNA species of low abundance. To increase the amount of probe available for screening, the cDNA probe was cloned and amplified. Two separate cDNA 'probe' libraries were constructed in the Escherichia coli plasmid vector pDE613, using poly(A)+mRNA from murine cells at 0 and 16 h after stimulation of a G0 population. Radiolabelled plasmid DNA from each library was hybridized sequentially to colony blots of the third 'target' library, constructed with mRNA from serum-stimulated cells in the Bacillus subtilis vector pBD214. Differential screening of the target cDNA library with the two probe libraries identified novel murine cDNA clones, some representing cytoplasmic poly(A)+RNA species of low (0.01%) abundance, accumulating after serum stimulation of a quiescent mouse embryo fibroblast population. One cDNA clone was found to correspond to mitochondrial 16S rRNA and a second was identified as the murine equivalent of previously described cDNA clones for the hamster 78-kDa glucose-regulated protein (GRP78) and the rat immunoglobulin heavy-chain-binding protein. GRP78 mRNA has not previously been recognized as a serum-inducible message.

Amino Acid Sequence↗
PubMed1987

Evidence that post-transcriptional changes in the expression of mitogen regulated protein accompany immortalization of mouse cells.

Primary mouse embryo fibroblasts passed in culture go through a period of declining growth rate and then after a period of minimal proliferation begin to grow again, generating, under appropriate conditions, an immortal 3T3 line. The 3T3 cells, but not the primary mouse embryo fibroblasts, are able to synthesize and to secrete mitogen regulated protein (MRP, also known as proliferin). We report here that the level at which the change in gene expression occurs during this spontaneous immortalization process is post-transcriptional, reflecting a change in either RNA processing or transcript stability. To our knowledge, this is the first report of an alteration at the post-transcriptional level of gene expression that accompanies immortalization.

Animals↗
PubMed1986

Immunological detection of cDNA clones encoding the uncoupling protein of brown adipose tissue: evidence for an antigenic determinant within the C-terminal eleven amino acids.

Poly(A)+RNA was isolated from brown adipose tissue of cold acclimated rats and a fraction enriched for uncoupling protein mRNA was used to generate a cDNA library in pBR 322. Immunological screening of 1,500 colonies with an affinity-purified antiserum against the uncoupling protein yielded five positive clones, pUCPrat1-5. Clone pUCPrat2 encoded the C-terminal 54 amino acids of rat uncoupling protein and exhibited 90% amino acid homology with the hamster protein. Clones pUCPrat3-5 encoded only the C-terminal 11 amino acids suggesting that an antigenic determinant lies within this sequence.

Adipose Tissue↗
PubMed1986

Close relationship of the major excreted protein of transformed murine fibroblasts to thiol-dependent cathepsins.

Complementary DNA clones corresponding to 638 nucleotides of the messenger RNA encoding the major portion of murine major excreted protein have been isolated and sequenced. The amino acid sequence of a part of the murine major excreted protein deduced from the DNA sequence reveals substantial and significant homology with the cysteine proteases actinidin, rat cathepsin H, and papain. Since the amount of murine major excreted protein secreted by cultured cells is often enhanced by transformation, it is implicated in oncogenic phenomena and may play a role in the metastatic process by virtue of its proteolytic activity.

Amino Acid Sequence↗
PubMed1985

A pBR322-derived vector for cloning blunt-ended cDNA: its use to detect molecular clones of low-abundance mRNAs.

In place of the unique Pst I site in pBR322, we have engineered by GC tailing a unique Sma I site bracketed by Pst I sites. The resulting vector, pDE61, and an improved derivative with greater symmetry around the Sma I site, pDE613, have been used to clone blunt-ended duplex cDNA molecules in Escherichia coli in an efficient manner (5 X 10(5) clones from 1 microgram of double-stranded cDNA). When DNA is cloned into the Sma I site, the ability of both vectors to confer ampicillin resistance is lost. Evidence suggests that functional beta-lactamase is made only after the GC-rich sequence containing the Sma I site is deleted: an insert in the Sma I site prevents this. Libraries in either vector, with single or multiple inserts, can be used to generate amplified amounts of cloned heterogeneous cDNA for screening other "target" libraries in a non-homologous vector (e.g., a Bacillus subtilis vector) for cDNA clones of low-abundance mRNAs. Species as infrequent as 0.003% can be readily detected by colony hybridization.

Cloning, Molecular↗
PubMed1985

Transcriptional regulation of two serum-induced RNAs in mouse fibroblasts: equivalence of one species to B2 repetitive elements.

We obtained eight cDNA clones that define five genes whose expression (appearance of transcripts in the cytoplasm) is enhanced when quiescent mouse fibroblasts are stimulated with serum to divide. Two of these clones (designated 49C8 and 16C8) correspond to RNA species that are present in the cytoplasm of quiescent cells at very low levels. After serum stimulation, the level of 16C8 mRNA rose more rapidly than that of 49C8 RNA, reaching a maximum around 6 to 12 h. The data suggest that 49C8 and 16C8 RNAs are induced as a result of independent stimuli. Either fibroblast growth factor or 12-tetradecanoylphorbol-13-acetate alone could induce 16C8 expression almost as effectively as serum; in contrast, 49C8 was not efficiently induced by epidermal growth factor, fibroblast growth factor, insulin, or 12-tetradecanoylphorbol-13-acetate. Inhibitors of transcription and translation diminished the induction of 16C8, while 49C8 expression was sensitive to actinomycin D but not cycloheximide or 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole. In vitro transcription experiments with isolated nuclei revealed a peak in transcriptional activity of the 16C8 gene at around 3 h after serum stimulation. Sequence analysis of the 49C8 cDNA clone showed greater than 90% homology of a large portion to a consensus rodent B2 repetitive element.

Animals↗
PubMed1985

Characterization of a cDNA clone encoding murine mitogen-regulated protein: regulation of mRNA levels in mortal and immortal cell lines.

Mitogen-regulated protein (MRP) is secreted by certain immortal murine cell lines (Swiss 3T3, BNL) stimulated with serum or particular growth factors. We have identified a cDNA clone that encodes part of the protein and have confirmed that MRP is closely related to, if not identical to, the prolactin-related protein designated proliferin. MRP is not produced by primary mouse embryo fibroblasts to nearly the same extent as it is produced by many immortal or transformed lines. Control of expression of this protein by growth factors is achieved both by regulating the extent of transcription and by regulating the processing of the protein.

Animals↗