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At least 19 recordsLinked to original sources
PubMed1984

Molecular phenotyping of non-transformed and chemically transformed C3H10T1/2 mouse fibroblasts.

A systematic molecular phenotyping approach based on two-dimensional gel electrophoresis is being applied in an attempt to identify protein changes associated with malignant transformation. Using the C3H10T1/2 mouse cell line, two-dimensional polypeptide maps of the non-transformed cell line, several chemically transformed lines and a tumour cell line were compared. Although there is a large degree of similarity between the protein profiles of all cell lines, clear differences are evident. Initial results are consistent with the view that many of the protein changes are incidental to malignant transformation. Changes induced by 3-methylcholanthrene are retained after transplantation of the cells into nude mice.

Animals↗
PubMed1988

Growth support and toxicity of homocysteine and its effects on methionine metabolism in non-transformed and chemically transformed C3H/10T1/2 cells.

The effects of homocysteine (Hcy) on one non-transformed (Cl 8) and two malignant clones (Cl 16 and Cl T422) of the C3H/10T1/2 mouse embryo fibroblasts, were examined with regard to toxicity, ability to support growth and effects on methionine (Met) metabolism and glutathione level. Homocysteine in its reduced form (Hcy-SH) was toxic to all cell lines, and the LD90 was estimated to be 1.0 X 10(-4) M for Cl 8 and Cl 16 cells measured by plating efficiency, 0.8 X 10(-4) M for Cl 8 and 0.3 X 10(-4) M for Cl 16 when measured by total cell growth. At toxic concentrations, Hcy-SH showed a drastic effect on cell morphology both in the presence and absence of Met. The same effect was demonstrated with L-cysteine. No toxic effect was seen with homocystine (Hcy-SS-Hcy) or homocysteine thiolactone (Hcy-tl) at similar concentrations. Hcy-tl supported growth of both the non-transformed and malignant cells in Met-deficient medium but with decreasing efficiency in the order Cl 8, Cl 16 and Cl T422. The growth rate constant compared to that of Met-supplemented medium was 0.62 for Cl 8, 0.44 for Cl 16 and 0.38 for Cl T422 cells. The intracellular level of S-adenosylhomocysteine (AdoHcy) increased in all three cell lines in Hcy-tl-supplemented medium. The S-adenosylmethionine (AdoMet) content increased in Cl 8 cells, was constant in Cl 16 cells and decreased in Cl T422 cells under the same conditions. This resulted in a constant ratio of AdoMet/AdoHcy in the non-transformed cells (Cl 8) whereas this ratio decreased by 40% in Cl 16 and by 72% in Cl T422 cells when Hcy-tl replaced Met in the medium. The ability of Hcy-tl to support growth thus seemed to correlate well with alteration in Met metabolism in this cell culture system. The intracellular level of glutathione (GSH) was measured during exponential growth, but showed small variations between non-transformed cells and Cl 16 cells. However, Cl T422 cells showed a distinct lower level of GSH in Met-supplemented medium, and this increased 3- to 4-fold when Met was replaced with Hcy-tl.

Animals↗
PubMed1984

Characterization of human transforming genes from chemically transformed, teratocarcinoma, and pancreatic carcinoma cell lines.

Dominant transforming genes that were transferred to mouse NIH3T3 cells by cellular DNAs prepared from a chemically transformed human cell line (MNNG-HOS), a human teratocarcinoma cell line (PA1), and a human pancreatic carcinoma cell line (A1165) were characterized (a) analyzing the repetitive human DNA sequences that were associated with the transforming gene and (b) determining their relationship to the oncogenes of the Harvey (rasH) and Kirsten (rasK) sarcoma viruses and to the human neuroblastoma transforming gene (rasN). The results show that the transforming gene activated in the teratocarcinoma cell line is identical to the neuroblastoma transforming gene and that the transforming gene of the pancreatic carcinoma cell line is a human homologue of rasK. In contrast, the transforming gene activated in the chemically transformed human cell line showed no detectable homology to rasK, rasH, and rasN.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1981

Transforming growth factor production by chemically transformed cells.

Evidence is presented indicating that the chemically transformed AKR-MCA and C3H/MCA-58 murine cell lines produce "transforming growth factor(s)" capable of inducing a transformed morphology and the ability to grow in soft agar in nontransformed, anchorage-dependent indicator cells. Serum-free medium conditioned by exposure to the chemically transformed cells was chromatographed on a Bio-Gel P-60 column after dialysis and lyophilization. Using the nontransformed mouse AKR-2B cells as the indicator cells, a peak of soft agar growth-stimulating activity was detected in the molecular weight range of 10,000 to 12,000. The soft agar growth-stimulating activity in pooled fractions from the AKR-MCA cells was shown to be trypsin and dithiothreitol sensitive and relatively heat stable; the activity was not destroyed by heating to 56 degrees for 30 min or to 100 degrees for 3 min. The pooled material also caused stimulation of growth in the soft agar of rat NRK cells and stimulation of DNA synthesis in the AKR-2B cells. The quantity required to give significant competition for binding to the epidermal growth factor receptor was about one order of magnitude greater than that required for stimulation of soft agar growth. Further separation of these polypeptide(s) by carboxymethylcellulose chromatography revealed three apparent peaks of soft agar growth-stimulating activity. Epidermal growth factor receptor-competing activity cochromatographed with the early minor soft agar growth-stimulating peak, whereas the two major peaks of soft agar growth-stimulating activity had no associated detectable competition for epidermal growth factor binding to its receptor. The data indicate that at least a major portion of the transforming growth factors produced by the chemically transformed cells is different from those described previously in murine sarcoma virus-transformed mouse cells and human tumor cells.

Animals↗
PubMed1980

Isolation of mutants temperature-sensitive for expression of the transformed state from chemically transformed C3H/10T1/2 cells.

58 MCA Cl 16 is an oncogenic methylcholanthrene-transformed variant of the non-transformed mouse embryo fibroblast cell line, C3H/10T1/2 Cl 8. Using two different protocols, we have isolated six temperature-sensitive mutants from N-methyl-N'-nitro-N-nitrosoguanidine treated cultures of 58 MCA Cl 16. C3H/10T1/2 Cl 8, 58 MCA Cl 16 and the six mutant lines were characterized with respect to several properties associated with the transformed state: morphology, saturation density, anchorage independence, cell surface morphology and growth in medium containing 1% fetal calf serum. In general, C3H/10T1/2 cells behaved as non-transformed, whether grown at 33 degrees C or 39.5 degrees C. The transformed parental line and all six mutants behaved as transformed cells at 33 degrees C. At 39.5 degrees C, only the parental transformed line retained the transformed phenotype. Three of the mutants revert towards non-transformed behavior at 39.5 degrees C for all of the properties tested. The remaining mutants are temperature-sensitive for some, but not all, transformed characteristics. Thus, while the expression of these transformed properties is sometimes coupled, we have been able to dissociate the expression of traits such as saturation density, anchorage independence and transformed morphology from each other. These mutants should prove to be valuable tools in the study of the mechanisms which underly the expression of the chemically-induced transformed state.

Animals↗
PubMed1980

Transforming activity of DNA of chemically transformed and normal cells.

DNA fragments of chemically transformed and normal avian and murine cells induce transformation of NIH 3T3 mouse cells with low efficiencies. High molecular weight DNAs of cells transformed by DNA fragments induce transformation with high efficiencies in secondary transfection assays. It thus seems that endogenous transforming genes of uninfected cells can be activated and efficiently transmitted by transfection. These results are consistent with the hypothesis that normal cells contain genes that are capable of inducing transformation if expressed at abnormal levels.

Animals↗
PubMed1988

Transforming growth factor alpha in chemically transformed hamster oral keratinocytes.

The cheek pouch of the Syrian hamster is an excellent tissue for the experimental induction of oral cancer by carcinogenic chemicals. Lysate prepared from a cell line (HCPC-1) derived from one of these hamster oral tumors greatly increased the growth of these oral tumor cells in vitro. We now show that the mitogenic substance, transforming growth factor alpha (TGF-alpha), is present in all of the chemically transformed hamster oral tumors examined (in vitro and in vivo). In no adult normal tissue of the Syrian hamster can we detect expression of TGF-alpha. TGF-alpha could be partly or wholly responsible for the mitogenic activity detected in the lysate of the chemically transformed hamster oral keratinocytes. Both normal and chemically transformed hamster oral keratinocytes express the receptor to epidermal growth factor. The consistent detection of TGF-alpha and epidermal growth factor receptor mRNAs in these hamster oral tumor cells suggests that an autocrine growth mechanism might be operative. This hamster cheek pouch oral cancer model can be used for the molecular analysis of how TGF-alpha and epidermal growth factor receptor might be involved in the malignant transformation of epithelial tissues.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1997

Aberrant O-glycosylation in the collagenous domain of pro alpha2(I) procollagen subunits synthesized by chemically transformed hamster fibroblasts.

Chemically transformed Syrian hamster embryo fibroblasts (NQT-SHE) do not synthesize the pro alpha1(I) subunit of type I collagen, but they secrete two forms of the pro alpha2(I) subunit (N33 and N50) with abnormal post-translational modifications localized in the alpha2CB3,5 cyanogen bromide peptide of the collagenous domain (B. Peterkofsky and W. Prather (1992) J. Biol. Chem. 267 5388-5395). Isoelectric focusing and treatment of the modified chains with glycosidases and biotinylated Jacalin lectin identified the modifications as Gal beta1,3-GalNAc-O-Ser/Thr with or without a terminal sialic acid in an alpha2,6 linkage. Unhydroxylated N33 alpha-chains also reacted with Jacalin, confirming that the abnormal modification was O-glycosylation and not hyperhydroxylation of proline or lysine. Cells were treated with benzyl GalNAc, a competitive inhibitor of galactosyl transferase that prevents addition of Gal to GalNAc-O-Ser/Thr and thus blocks elongation of O-glycosyl chains. Treated cells secreted pro alpha2(I) chains containing GalNAc-O-Ser/Thr but no galactose or sialic acid, which suggested that Gal addition takes place before sialylation. Treatment of NQT-SHE cells with monensin and brefeldin A inhibited secretion and led to intracellular accumulation of pro alpha2(I) chains that contained only GalNAc. Therefore, it appears that GalNAc addition to pro alpha2(I) chains in NQT-SHE cells occurs in the cis-Golgi, while sialic acid and galactose are added in the trans-Golgi network. The pro alpha2(I) chains produced by NQT-SHE cells most likely are modified because they are in the denatured state, and thus potential O-glycosylation sites become available that would not be exposed in normal triple helical procollagen.

Acetylgalactosamine↗
PubMed1979

Study of the avian sarcoma virus infection of chemically transformed cells.

BHK-21/13 cells transformed with various chemical carcinogens and mutagens were tested for susceptibility to avian sarcoma virus infection. The chemically transformed BHK cells were highly tumorigenic. The treatment of these cells in vitro with avian sarcoma virus strain Schmidt-Ruppin D resulted in chemically transformed and viral infected cells with different rescuability of the integrated virus genome. The different rescuability is not due to the difference in virus penetration as was shown by vesicular stomatitis virus Schmidt-Ruppin virus pseudotype VSV(SR-D) technique. The sarcoma virus genome in the doubly transformed cells was not inducible by various virus inducers.

Animals↗
PubMed1983

Induction of reverse transformation and normal cell cycle regulation by dibutyryl cAMP in a chemically transformed cell line.

The objective of this study was to determine whether N6, O2-dibutyryl 3',5'-adenosine monophosphate (db-cAMP)-induced reverse transformation in a chemically transformed mouse cell line, AKR-MCA, would restore normal cell cycle regulation, particularly with regard to their growth arrest in the early G1 period. The AKR-MCA cells were grown to confluency in the presence or absence of db-cAMP (0.5 mM) plus theophylline (1 mM). The confluent cultures were trypsinized and a portion of the cells were fused with mitotic HeLa cells to induce premature chromosome condensation, while the remaining cells were used to study the kinetics of initiation of DNA synthesis. The prematurely condensed chromosomes (PCC) of the control and the treated cultures were classified into G1, S, or G2 types on the basis of their morphology. The G1 PCC were further subclassified into six groups (+1 - +6); +1 being the most condensed and +6 the most decondensed. The cyclic AMP (cAMP)-treated cells exhibited better attachment to the culture dish, were blocked in early G1 period at confluency, and entered S phase about 4 h later than the control following subculturing. In contrast, a majority of cells in the control cultures were arrested in S phase at confluency. These data indicate that the db-cAMP-induced reverse transformation in AKR-MCA cells at least partially restores normal cell cycle regulation in these chemically transformed cells.

Animals↗
PubMed1983

Comparison of intra- and extracellular transforming growth factors from nontransformed and chemically transformed mouse embryo cells.

Multiple transforming growth factors (TGFs) have been isolated from the serum-free conditioned media and acid-ethanol cell extracts of nontransformed AKR-2B and chemically transformed AKR-MCA mouse cells. The TGF activity was analyzed using Bio-Gel P-60 chromatography in 1 M acetic acid and tested for colony-stimulating activity in soft agar using AKR-2B, AKR-2B (clone 84A), NRK, and AKR-MCA cells as indicators. Both intracellular and extracellular TGF activity from AKR-MCA and AKR-2B cells show a major peak of AKR-2B and epidermal growth factor-potentiated NRK colony-stimulating activity that coelute in the 13,000 +/- 2,000 molecular weight region. In the 24,000 +/- 7,000 molecular weight range, AKR-MCA cells produce intracellular and extracellular NRK colony-stimulating activity that is not potentiated by epidermal growth factor, while the intracellular and extracellular NRK colony-stimulating activity produced by AKR-2B cells requires added epidermal growth factor for colony formation. Also important in determining the growth and morphological characteristics of a cell line, besides the production of a TGF, is the ability of a cell to respond to TGF activity. We have shown that the transformed AKR-MCA cells form more colonies than AKR-2B cells in response to certain TGF activities. This suggests that the increased responsiveness of AKR-MCA cells to TGFs may be important in determining its phenotype.

Animals↗
PubMed1989

Regulation of transforming growth factor alpha messenger RNA expression in a chemically transformed rat hepatic epithelial cell line by phorbol ester and hormones.

Transforming growth factor alpha (TGF-alpha) is produced by many transformed cells, but little is known about the regulation of its expression. We examined TGF-alpha mRNA levels in a set of cloned neoplastic cell lines derived by chemical transformation of a normal rat liver epithelial cell. The untransformed parental cell line, WB-344, did not express a detectable level of TGF-alpha mRNA, whereas GP6ac, a transformed line capable of autonomous growth in soft agar, expressed TGF-alpha. When GP6ac cells were treated with agents thought to regulate protein kinase C activity, e.g., the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA), TGF-alpha mRNA levels increased by 8- to 11-fold. The induction of TGF-alpha mRNA was detectable at 2 h, was maximal at 8-12 h, and declined by 24 h. Angiotensin, bradykinin, epinephrine, and epidermal growth factor also increased TGF-alpha mRNA by 2- to 5-fold. In contrast, parental WB cells neither expressed TGF-alpha mRNA, nor responded to TPA. TPA also increased epidermal growth factor receptor mRNA in GP6ac cells but the effect was less prolonged; maximal levels were seen at 4 h after TPA exposure and returned to control levels by 12 h. TPA increased TGF-alpha mRNA in GP6ac cells, in part, by increasing transcription of the TGF-alpha gene as measured by run-on transcription rates in isolated nuclei. In addition, the induction of TGF-alpha by TPA was blocked by concurrent incubation with agents that inhibit protein synthesis. However, if TPA was present for at least 2 h, subsequent addition of cycloheximide enhanced the effect of TPA. This indicates that the induction of TGF-alpha in GP6ac cells is comprised of at least two phases demarcated by the requirement for protein synthesis. The time course of induction and the sensitivity to inhibition of protein synthesis distinguish the effect of TPA on TGF-alpha mRNA from that of other genes regulated by TPA, e.g., c-myc and c-fos. These data also suggest that chemical transformation of rat liver epithelial cells leads to expression of TGF-alpha mRNA, and that once expressed, TGF-alpha mRNA can be modulated in a protein kinase C-dependent manner.

Animals↗
PubMed1982

Growth factors, growth factor receptors, and cell cycle control mechanisms in chemically transformed cells.

Previous studies have shown that the chemically transformed AKR-MCA and C3H/MCA-58 cell lines spontaneously arrested growth at high saturation density in the G1 phase of the cell cycle because of depletion of low-molecular-weight nutrients (amino acids and glucose) from the medium. The nontransformed parent cell lines, AKR-2B and C3H/10T1/2, spontaneously arrest growth in G1 at low saturation density because of depletion of essential serum growth factors. If prevented from becoming deficient in growth factor by maintenance in medium with mitogens such as epidermal growth factor (EGF), fibroblast growth factor, or the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, the nontransformed AKR-2B cells behave similarly to the transformed cells and arrest growth in G1 at high saturation density because of nutrient deficiency. This suggests that a major difference between the nontransformed and chemically transformed cells is an inability of the latter to achieve growth factor deficiency. In addition, the transformed AKR-MCA and C3H/MCA-58 cells show greatly diminished detectable EGF receptors. These observations could be accounted for by the endogenous production of the response to a growth factor-like substance by the transformed cells. Preliminary data indicate that these chemically transformed cells release a transforming growth factor (TGF) into serum-free medium. Whether TGF is similar or identical to the previously described sarcoma growth factor remains to be established.

Cell Cycle↗
PubMed1994

"Libraries from libraries": chemical transformation of combinatorial libraries to extend the range and repertoire of chemical diversity.

The generation of diverse chemical libraries using a "libraries from libraries" concept is described. The central features of the approaches presented are the use of well-established solid-phase synthesis methods for the generation of combinatorial libraries, combined with the chemical transformation of such libraries while they remain attached to the solid support. The chemical libraries that are generated by this process have very different physical, chemical, and biological properties compared to the libraries from which they were derived. A wide range of chemical transformations are possible for peptide-based or other libraries, and an almost unlimited range of useful chemical diversities can be envisioned. In the example presented, the amide functionalities in an existing combinatorial library made up of peptides were permethylated while the library remained attached to the solid-phase support used in its synthesis. After removal of the permethylated mixtures from their solid support, this library, now lacking the typical -CONH- amide bonds of peptides, can be tested in solution with virtually all existing assay systems to identify individual compounds having specific biological activities of interest. An illustration of the use of such libraries is presented, in which the described permethylated library was used to identify individual permethylated compounds having potent antimicrobial activity against Gram-positive bacteria.

Amino Acid Sequence↗
PubMed1999

Metastatic conversion of chemically transformed human cells.

A linear model for human cell metastasis has been developed in vitro from chemically transformed normal human cells. The chemically transformed cells are nontumorigenic in nude mice, but can be converted to a tumorigenic phenotype by transfection with a nondirectional cDNA library or antisense cDNA to the ML-1 gene. The primary transfected cell line (TR1T) forms localized, progressively growing tumors in nude mice that do not invade into the surrounding tissue. This tumorigenic TR1T cell line could be advanced into a metastatic stage following an additional transfection (TR2M cell line) with the cDNA expression library or antisense cDNA to the ML-1 gene. Metastatic cells, selected from tumors that were attached to internal organs, exhibited an increase in invasiveness as measured in vitro using an invasion chamber. The metastatic cells also exhibited an increased expression of matrix metalloproteinase-1 (MMP-1), although MMP-1 was not part of the cDNA that was transfected into either the TR1T cells or the doubly transfected metastatic TR2M cells. These data suggest that the increase in MMP-1 expression was a secondary downstream event responding to an upstream genetic change that initiated the conversion of cells from a tumorigenic to a metastatic stage. In summary, human cell lines representing premalignant, malignant, and metastatic phenotypes have been established in culture that can be used to identify gene changes that occur as normal human cells progress to a metastatic stage during tumor development. One gene, ML-1, that is found in the expression library appears to be involved in malignant progression, because ML-1 antisense cDNA will convert chemically transformed cells to both tumorigenic and metastatic stages, and cells from both local and metastatic tumors have a reduced or complete loss of expression of the ML-1 gene.

Animals↗