Joseph R. Landolph

Molecular and cellular mechanisms of transformation of C3H/10T1/2 Cl 8 and diploid human fibroblasts by unique carcinogenic, nonmutagenic metal compounds: A review

Work from our laboratory showed that carcinogenic metal salts of arsenic, nickel, and chromium induced morphological transformation of cultured C3H/10T1/2 Cl 8 (10T1/2) mouse embryo cells, and that many of the transformants grow in soft agarose and form tumors in nude mice. Concentrations of arsenic, nickel, and chromium compounds that induced morphological transformation did not induce mutation to ouabain resistance in 10T1/2 cells. This indicated that the mechanism of metal induced morphological transformation was likely not caused by induction of base substitution mutations, and in the case of lead chromate, likely not caused by frameshift or deletion mutations.In addition, we showed that carcinogenic arsenic, nickel, and chromium compounds, and MNNG, induced anchorage independence in diploid human fibroblasts. Anchorage-independent cell strains derived from anchorage-independent colonies were stable but did not form foci and eventually senesced, therefore, arsenic and nickel compounds and lead chromate induced stable anchorage independence as an isolated phenotype. Nickel compounds and lead chromate induced anchorage independence but not mutation to ouabain resistance or to 6-thioguanine resistance. Hence, the mechanism of induction of anchorage independence by these metal salts in human fibroblasts was likely not via induction of base substitution, frameshift, or deletion mutations that would be measured in these mutation assays. MNNG, on the other hand, induced mutation to 6-thioguanine resistance and to ouabain resistance over the same concentration ranges that induced anchorage independence was induced, indicating that MNNG might induce anchorage independence by inducing base substitution, frameshift, or deletion mutations. It is likely, therefore that the mechanisms of metal salt induced morphological and anchorage independent transformation in murine and human base substitution mutations. We hypothesize that rearrangements or amplification of proto-oncogenes, or, possibly, inactivation of suppressoro oncogenes, might play a role in the mechanism of metal salt induced morphological or anchorage independent transformation of 10T1/2 and diploid human fibroblasts, respectively. *** DIRECT SUPPORT *** A03GS019 00027

DT-diaphorase activity and the cytotoxicity of quinones in C3H/10T1/2 mouse embryo cells

A permanent mouse fibroblast cell line derived from C3H mouse embryos, C3H/10T1/2 C18, was used to study the cytotoxicity of some model quinones under conditions in which DT-diaphorase (EC 1.6.99.2) activity was induced or inhibited. Sudan III [1-[[4-(phenylazo)phenyl]azo]-2-naphthalenol] and 3-methylcholanthrene (MCA), but not butylated hydroxyanisole (BHA), induced DT-diaphorase in a concentration-dependent manner. Induction of DT-diaphorase activity was dependent upon new RNA and protein synthesis, as shown by experiments employing actinomycin D and cycloheximide respectively. Induction of DT-diaphorase by Sudan III or MCA was associated with protection against the cytotoxicity of quinones as measured by a colony survival assay. When control and induced cells were also exposed to dicoumarol, a specific and potent inhibitor of DT-diaphorase, the cytotoxicity of the quinones in both control and induced cells was enhanced markedly. The results support the hypothesis that DT-diaphorase competes with one-electron quinone-reducing enzymes (such as cytochrome P-450 reductase) which generate auto-oxidizable semiquinones and forms more stable hydroquinones as an initial step in the detoxification of quinones in 10T1/2 cells.

Molecular biology of nickel carcinogenesis: Identification of differentially expressed genes in morphologically transformed C3H10T1/2 Cl 8 mouse embryo fibroblast cell lines induced by pecific insoluble nickel compounds

Inhalation of mixtures of insoluble and soluble nickel compounds by humans during nickel refining has been associated with excess lung and nasal sinus cancers. Insoluble nickel subsulfide (Ni3S2) and nickel oxide (NiO) are carcinogenic to rodents by inhalation. We previously showed that insoluble Ni3S2, crystalline nickel monosulfide (NiS), and green (high temperature, HT) and black (low temperature, LT) NiO, induced morphological transformation in cultured C3H/10T1/2 Cl 8 (10T1/2) mouse embryo cells. To understand molecular mechanisms of carcinogenesis by insoluble nickel compounds, we used random, arbitrarily primed-polymerase chain reaction (RAP-PCR) mRNA differential display and identified nine cDNA fragments that were differentially expressed between nontransformed and nickel-transformed cell lines in ~ 10.0% of the total mRNA. Expression of the calnexin gene (encoding a type I membrane protein/molecular chaperone), the ect-2 proto-oncogene, and the stress-inducible gene, Wdr1, was upregulated. Expression of six genes – the vitamin D interacting protein/thyroid hormone activating protein 80 (DRIP/TRAP-80) gene, the insulin-like growth factor receptor 1 (IGFR1) gene, the small nuclear activating protein (SNAP C3) gene, and three unknown genes, was down-regulated, in nickel-transformed cell lines. We hypothesize that these resulting aberrations in gene expression could contribute to the induction and/or maintenance of morphological transformation induced by specific insoluble nickel compounds.

S-9 Metabolic activation enhances aflatoxin-mediated transformation of C3H/10T 1/2 cells

The use of a rat liver S-9 metabolic activation system to enhance aflatoxin B1-mediated transformation of C3H/10T1/2 cells was studied. Under conditions of metabolic activation, the cytotoxicity of Aflatoxin B1 (AFB) was increased approximately 10-fold over that observed in the absence of activation. Similarly, activation increased the transformation of these cells treated with 1 microgram/ml AFB1 greater than 10-fold when cells were treated in the absence of activation with 1 microgram/ml AFB1 for 3 hr, and four-to-fivefold over the transformation observed when cells were treated for 48 hr with 1 microgram/ml AFB1. This same activation procedure also induced the transformation of these cells treated with 10 and 20 micrograms/ml cyclophosphamide in the absence of activation. The use of S-9 metabolic activation greatly increased the sensitivity of AFB1-mediated transformation of C3H/10T1/2 cells, and should expand the range of chemical carcinogens, requiring metabolic activation, in particular mycotoxins related to AFB1, that can be effectively detected and studied in the C3H/10T1/2 cell transformation assay.

Soluble vs insoluble hexavalent chromate. Relationship of mutation to in vitro transformation and particle uptake.

Soluble CaCrO4 and insoluble PbCrO4 were tested for induction of mutation to 6-thioguanine (base-substitution, deletion, addition, and frameshift mutations) or ouabain (base-substitution mutations) resistance in Chinese hamster ovary cells and morphological transformation in C3H/101/2 mouse embryo cells. CaCrO4 induced dose-dependent cytotoxicity and mutation to 6-thioguanine resistance, but did not induce mutation to ouabain resistance or morphological transformation. Highly cytotoxic amounts of CaCrO4 induced conversion of 10T1/2 cells to adipocytes, but cell lines derived from such cells were not transformed. PbCrO4 was not mutagenic in either mutation assay but induced a dose-dependent, low frequency of focus formation. Cell lines established from these foci had a 3–5-fold increased saturation density, grew in soft agarose, and were tumorigenic in nude mice. Chronic exposure to CaCrO4 or PbCl2 did not induce transformation, PbCl2 was inactive even at acutely cytotoxic concentrations, and sequential treatments with CaCrO4 and PbCl2 did not induce transformation. Light and scanning electron microscopy showed progressive cytoplasmic engulfment of PbCrO4 particles and extensive vacuolization of cells in contact with the particles. No particles were observed inside of vacuoles. We suggest that internalization of PbCrO4 and the associated cellular stress response may be related to PbCrO4-induced neoplastic transformation of 10T1/2 cells.

Further evidence that ouabain-resistant variants induced by chemical carcinogens in transformable C3H/10T1/2 Cl 8 mouse fibroblasts are mutants

Abstract Ouabain-resistant (Oua r ) variants were induced in C3H/10T1/2 Cl 8 cells by the chemical carcinogens, N -methyl- N ′-nitro- N -nitrosoguanidine (MNNG), N -acetoxy- N -2-acetylaminofluorene (N-AcO-AAF), and benzo[ a ]pyrene (BaP). The use of the Poisson calculation to determine Oua r variant frequencies gave more linear dose-response curves than when variant frequencies were calculated from the observed number of Oua r colonies. Increasing the Oua concentration from 3 to 6 mM decreased the frequency of Oua r variants. When cloned Oua r variants were mixed with wild-type cells, there was no metabolic cooperation and no loss of mutants when mock expression-time curves were determined. Oua r variants remained Oua r after prolonged cultivation in the absence of Oua. 86 Rubidium ( 86 Rb) uptake was at least 10-fold more resistant to inhibition by Oua in Oua r variants than in wild-type cells. In one Oua r clone, one-third of the 86 Rb uptake was not inhibited by Oua concentrations as high as 10 mM, indicating that C3H/10T1/2 Cl 8 cells might be triploid at the Oua r locus. The relationship between the inhibition of 86 Rb uptake and the cytotoxicity caused by the same concentration of Oua was the same for 2 Oua r clones and wild-type C3H/10T1/2 Cl 8 cells. Therefore, the Oua r variants detected by this assay are most likely true mutants possessing an altered Na + K + transport system, the Na + K + Mg 2+ -activated adenosine triphosphate (ATPase), that is more resistant to Oua inhibition than the ATPase in wild-type cells.

Microcell-mediated transfer of carcinogen-induced ouabain resistance from c3h/10t1/2 cl 8 mouse fibroblasts to human cells.

We previously developed a quantitative assay for measuring the induction of ouabain-resistant (Ouar) variants in transformable C3H/20T1/2 Cl 8 mouse fibroblasts following treatment of the cells with chemical carcinogens. To further define the nature of the Ouar phenotype, we conducted microcell-mediated chromosome transfer studies using Ouar cell lines induced by chemical carcinogens in C3H/10T1/2 Cl 8 cells as donors and 8-azaguanine-resistant (Azgr) derivatives of the human cell lines, D98/AH2 and HT 1080, as recipients. Microcells prepared from one spontaneous and two carcinogen-induced Ouar mouse cell lines were able to transfer resistance to 0.01 and 1 mM Oua to ouabain-sensitive D98 and HT 1080 cells. The frequency of microcell hybrid formation ranged from 10(-6) to 10(-5). Karyotypic analysis of the microcell hybrids indicated that the Ouar phenotype of C3H/10T1/2 Cl 8 derivatives mapped to mouse chromosome 3, the chromosome to which the wild-type murine Oua-1 allele had previously been assigned. These studies show that both spontaneous and chemically induced high level Ouar phenotypes of C3H/10T1/2 Cl 8 mouse fibroblasts can be transferred via microcell-mediated chromosome transfer, and provide strong genetic evidence that chemically induced Ouar phenotypes of C3H/10T1/2 Cl 8 cells arise from mutations at Oua-1. In addition, this study sufficiently standardizes microcell-mediated chromosome transfer in the C3H/10T1/2 Cl 8 cell line so that this technique can be used to investigate the nature of other phenotypic changes in these cells, such as the chemically transformed phenotype.

Diabetes and Cancer: Epidemiological, Clinical, and Experimental Perspectives

Diabetes is a major cause of death in many countries due to its increasing incidence, high prevalence, and clinical manifestation of a variety of micro- and macrovascular complications if it is not appropriately treated [1, 2]. Recent studies have shown that diabetic patients may also have a higher risk of cancer [2], the number one killer that threatens the lives of billions of people.

Genetic and Probability Aspects of Cell Transformation by Chemical Carcinogens

Publisher Summary This chapter discusses the genetic and probability aspects of cell transformation by chemical carcinogens. In C3H/lOT1/2 cell system with 3-methylcholanthrene as the carcinogen, nothing, including replating, affects the validity of the probabilistic equations when n is greater than 5. The peak in the transformation curve at n = 4 to 5 cannot per se explain the high transformation frequencies obtained at low cell numbers. Possible explanations for this paradox might be: (1) that more than one gene mutation is required for transformation; (2) that the event resulting in transformation is not a simple gene mutation; or (3) that another nonmutational process requiring cell divisions is involved in transformation. Such a process might be categorized as “promotion” or possibly the production of chromosomal aberrations.

Abstract 1579: Induction of cytotoxicity by soluble chromium (VI) compounds in cultured C3H/10T1/2 Cl 8 mouse embryo cells effects of ascorbate and dehydroascorbate

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Hexavalent chromium [Cr(VI)]-containing compounds are human carcinogens. They cause cancers in the respiratory system when inhaled, and stomach/other internal cancers when ingested. Soluble and insoluble hexavalent chromium [Cr(VI)] compounds induced base substitution, deletion, addition, and frameshift mutations. Cr(VI) compounds also induce DNA-DNA cross links and DNA-protein cross-links in mammalian cells. We examined the ability of the soluble chromium compounds, sodium chromate (Na2CrO4), calcium chromate (CaCrO4) and potassium chromate (K2CrO4) to induce cytotoxicity and morphological transformation in C3H/10T½ Cl 8 (10T1/2) mouse embryo cells. We tested the hypothesis that the intracellular reductants, ascorbate and dehydroascorbate, can reduce Cr(VI) to Cr(V), Cr(IV), and Cr(III) intracellularly, making it a strong cytotoxin in mammalian cells. Ascorbate is present in serum at concentrations in the mM range under physiological conditions in humans, but is only present in the μM range in mouse embryo cells grown in BME cell culture medium plus 10% fetal calf serum. We hypothesized the relatively weak responses for induction of morphological transformation of mammalian cells by Cr(VI) compounds in culture (dose-dependent but weak induction of foci by lead chromate, and no induction of foci by calcium chromate, potassium chromate, and sodium chromate) is due to the small amounts of ascorbate in cultures of mammalian cells that are insufficient to reduce Cr(VI) to Cr(V), Cr(IV), and Cr(III). Therefore, we investigated the cytotoxic effects of ascorbate on 10T½ mouse embryo cells, and the effects of the highest non-cytotoxic concentrations of ascorbate on Cr(VI)-induced cytotoxicity in 10T½ cells, using reduction in plating efficiency as our cytotoxicity assay. Cell survival data showed that ascorbate itself exerted significant cytotoxic effects at concentrations of 0.00625 mM and higher on 10T½ mouse embryo cells. Furthermore, when 10T½ cells were treated with both Cr(VI) and ascorbate, ascorbate played dual roles. It served as a pro-oxidant (enhancer of the cytotoxicity of chromate) at concentrations up to 0.1 mM, and as an anti-oxidant (reducer of the cytotoxicity of chromate) at concentrations of 0.25 mM and higher. This information is important for our ongoing and future experiments, where we designed a protocol to incorporate ascorbate into our assays assessing the cytotoxicity and cell transforming activity of Cr(VI) compounds. We project that at concentrations of 0.1 mM and lower, ascorbate will enhance the cell transforming activity of Cr(VI) compounds. Supported by undergraduate fellowships from the Provosts Office at the University of Southern California (USC) (P. I., JRL), by funding from the M. S. Program in Molecular Microbiology and Immunology to JRL, by discretionary funding to JRL, and by prior grant ES03341 from NIEHS/NIH (P. I., JRL). Citation Format: Sophia A. Shahin, William Liao, Laureen Tran, Qasim A. Akinwumi, Farn-shuan Tseng, Alyssa Mathew-Joseph, Joseph R. Landolph. Induction of cytotoxicity by soluble chromium (VI) compounds in cultured C3H/10T1/2 Cl 8 mouse embryo cells effects of ascorbate and dehydroascorbate. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1579. doi:10.1158/1538-7445.AM2014-1579