Induction of morphological transformation by coal-dust extract in BALB/3T3 A31-1-13 cell line

The transforming activity of coal dust extracts was studied using BALB/3T3 clone A31-1-13 cells. Coal-dust extracts, both nitrosated and nonnitrosated, induced cell transformation in a dose-response manner. However, the transformation frequency was higher with the nitrosated than with the nonnitrosated extract. All transformed cell lines derived from coal-dust extract-induced foci showed biological characteristics of neoplastic transformation such as loss of contact inhibition and anchorage-independent growth. These results appear to support a hypothesis of coal mine dust causation of gastric cancer in coal miners.     

Activation of the Ha-ras gene in C3H 10T1/2 cells transformed by exposure to N-methyl-N'-nitro-N-nitrosoguanidine

A transfectable, presumably mutationally activated, c-Ha-ras gene was identified in a clonal population of 10T1/2 cells established from a Type II focus induced by exposure of a parental, wild-type population to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG).     

Effects of split-dose irradiation on survival and oncogenic transformation induced by 31 MeV protons in C3H10T1/2 cells

Survival and oncogenic transformation were studied in C3H10T1/2 cells exposed to 31 MeV protons. Total doses of 0.5, 1 and 7 Gy were delivered as single and two equal fractions with various time intervals up to 10 h between doses. With split doses as compared with single doses to a total dose of 7 Gy, survival increased by a factor of 2.5 +/- 0.2, whereas the frequency of transformation per surviving cell declined by a factor of 3.1 +/- 0.5. Maximal split-dose recovery occurred within the first 5 h for both endpoints. Further, the transformation frequency decreased by factors of 3.1 +/- 0.6 and 1.5 +/- 0.3 respectively for total doses of 0.5 and 1.0 Gy split into two equal fractions. The data for 1 and 7 Gy are compatible with data in the literature for other low LET radiations.     

Protective role of potentially lethal damage repair in the neoplastic transformation of Balb/c 3T3 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine

To determine the role of repair of potentially lethal damage (PLD) in the initiation process of neoplastic transformation, Balb/c 3T3 cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) were temporarily exposed to conditioned medium obtained from density-inhibited Chinese hamster cell cultures, as a post-treatment for the induction of PLD repair. With or without this exposure, cell survival and transformation frequencies were simultaneously determined by colony-formation and focus-formation assays, respectively. Temporary exposure to conditioned medium resulted in a 20-30% increase in cell survival compared with no exposure. Post-treatment with conditioned medium resulted in a 60-65% reduction in transformation frequencies. At the molecular level, the repair of MNNG-induced single-strand breaks of DNA occurred much more rapidly in conditioned medium. These data suggest that PLD repair reduces the in vitro neoplastic transformation through excision repair operative during the cessation of DNA replication. Thus, PLD repair appears to be preventive against neoplastic fixation in initiation of neoplastic development.     

p-Nonylphenol acts as a promoter in the BALB/3T3 cell transformation

p-Nonylphenol (NP) has attracted attention as an estrogenic contaminant, and the environmental pollution by NP has been found to be extensive. NP is classified as a phenolic antioxidant based on the chemical activity and structure. Some phenolic antioxidants are known to induce and/or enhance carcinogenesis. We examined the effects of NP on the two-stage transformation of BALB/3T3 cells, a model of two-stage carcinogenesis. The treatment by NP in the promotion phase markedly enhanced the transformation of the cells pre-treated with a subthreshold dose of a carcinogen, 3-methylcholanthrene (MCA), but not that of non-pretreated cells. The promoting activity of NP was approximately one hundredth of that of 12-O-tetradecanoylphorbol-13-acetate (TPA), a potent tumor promoter, in the cell transformation. The treatment by NP in the initiation phase did not induce cell transformation with and without post-treatment by TPA. These results indicate that NP acts as a pure promoter of cell transformation implying that it may cause the enhancement of carcinogenesis in vivo. The enhancement by NP of MCA-initiated transformation was suggested not to be mediated by estrogen receptors in BALB/3T3 cells because 17 beta-estradiol did not promote cell transformation in our experiments, and it has been reported that BALB/3T3 cells do not express estrogen receptors at a detectable level.     

Effect of Clostridium perfringens-derived wound healing substance as compared with epidermal growth factor on the growth and morphological transformation of BALB/3T3 A31-1-1 cells

Clostridium perfringens-derived wound-healing substance (WHS), having growth-stimulating activity, was examined to determine its effect on the growth and morphological transformation of BALB/3T3 A31-1-1 cells. WHS accelerated the cell growth at the exponential growth phase, shortening the doubling time by 8–18%. The maximum cell density of the treated cultures was slightly higher than that of the control culture, and the cell number decreased in the same way as the control cells did. On the other hand, the cells treated with epidermal growth factor (EGF) or insulin showed growth rates similar to that of the control cells during the exponential growth phase, and after the control cells attained the maximum cell number, the number of the treated cells continued to increase gradually for more than 4 days and then decreased. Under the experimental conditions of the two-stage transformation assay, application of WHS at the tumor-initiation or promotion stage did not accelerate the formation of transformed foci. Although treatment with EGF at the initiation stage induced no enhancement, marked enhancement of morphological transformation was observed in the treatment at the promotion stage. These results indicate that the mode of action between WHS and EGF or insulin is different on the growth-stimulating activity and morphological transformation of BALB/3T3 A31-1-1 cells.     

Altered kinase C function in transformed BALB/c3T3 cells

Comparative studies of kinase C function were performed in an untransformed (A31) and the benzo[a]pyrene (BPA31), dimethylbenz[a]anthracene (DA31), and Kirsten sarcoma virus (KA31) transformed BALB/c 3T3 mouse fibroblast cell lines. The 80-kDa kinase C dependent phosphoprotein (pp80), an in vivo marker of kinase C activity, was markedly decreased in the transformed cells although the amount of the 80-kDa substrate protein in the BPA31 cells was similar to that in the untransformed A31 cells. Total cell lysate kinase C levels were lower in the transformed cells but this difference could not account for the reduced pp80 phosphorylation. Increased affinity of kinase C for the membrane fraction in the BPA31 cells may account for decreased phosphorylation of pp80.     

Inhibition of BALB/c-3T3 cells in late G1: Commitment to DNA synthesis controlled by somatomedin C

Methylglyoxal bis-(guanylhydrazone) (mGBG) blocked the stimulation of DNA synthesis in quiescent, density-inhibited BALB/c-3T3 cells treated with platelet-derived growth factor (PDGF) and platelet-poor plasma (PPP). Competence formation produced by a transient exposure to PDGF was not effected by mGBG. In contrast, mGBG effectively inhibited the PPP-stimulated progression of competent cells through the G₁ phase of the cell cycle, although maximal inhibition was observed when mGBG was present during both the exposure to PDGF- and PPP-supplemented media. When quiescent cells were treated with PDGF and PPP-supplemented media in the presence of mGBG for 12–18 hours and the mGBG was then removed, cells entered the S phase after a 4 hour lag. The rate of entry into the S phase, but not the time necessary for the cells to progress from the mGBG block into the S phase, was dependent on the concentration of PPP present after removal of the mGBG. Either somatomedin C or insulin, but not epidermal growth factor, fibroblast growth factor, or PDGF were able to substitute for PPP in allowing cells to enter the S phase after the cells were released from the mGBG block. A marked inhibition of (³H)-leucine incorporation in serum-stimulated cultures was produced at mGBG concentrations which caused no decrease in the amount of (³H)-uridine incorporated during a short (15 minute) pulse. The ability of hormones to allow cells to progress to the late G₁ phase and become committed to DNA synthesis after a mGBG inhibition was not related to their ability to restore the normal rate of protein synthesis as determined by (³H)-leucine incorporation.     

Morphological transformation of C3H/10T1/2 cells subcultured at low cell densities

Morphological transformation in $\text{C3H/10T}\text{1}\text{2}$ cells treated with varied concentrations of benzo (α) pyrene (BP) was measured following subculture at low cell densities. Subconfluent cultures exposed to BP were allowed to grow to confluence, trypsinized, and reseeded at cell densities ranging from 5 to 2,300 surviving cells/cm². These secondary cultures were incubated for 8 to 9 weeks, stained, and examined for evidence of morphological transformation. BP-treated cells reseeded in virtual isolation in microwells (approx. 5 surviving cells/cm²) transformed at frequencies up to 14.5%. At these low initial cell densities, transformation frequency did not demonstrate a significant dependence on BP concentration. However, BP-treated cells reseeded at higher densities (11 to 2,300 surviving cells/cm²) showed both density-dependent transformation frequencies and BP-concentration dependence of transformation. As reported previously (Haber et al., Cancer Res. 37 1644, 1977), the subculturing of treated cells did not affect the BP-concentration dependence of focus formation in the $\text{C3H/10T}\text{1}\text{2}$ transformation assay. Cell density-dependent suppression of morphological transformation has now been observed over a wide range of BP concentration. We suggest that this phenomenon is associated with colony interactions and consider various possible mechanisms of BP involvement.     

Lactate-mediated changes in growth morphology and transformation frequency of irradiated C3H 10T1/2 cells

Treatment of mammalian cells with lactate or inhibitors of glycolysis alters their radiation response, particularly in the low dose region of the dose response curve. The occurrence of both high lactate levels and high glycolytic metabolism in tumours is well known and therefore the effect of lactate on a cell line sensitive to radiation induced transformation was examined using a single exposure to Cobalt 60 gamma rays as the carcinogen challenge. The results indicate that cells treated with 5mM lactate before irradiation exhibit changes in morphology and growth rate and that the transformation frequency is increased by three to ten fold following 24 hours lactate treatment just prior to irradiation. Examination of radiation survival curves showed a positive correlation between transformation frequency and size of the shoulder, but increasing transformation frequency was associated with a decrease in Do. A mechanism involving altered Redox potential in lactate treated cells is suggested. The results are discussed in terms of their possible significance for radiotherapy.     

Induction of malignant transformation by various chemicals in Balb/3T3 clone A31-1-1 cells and biological characterization of some transformants

Balb/c A31-1-1 cells were used for the study of transformation induction by chemicals with different mutagenic specificities. We show that survival of these cells and therefore the calculated transformation frequency per cells at risk is dependent upon the cell density at the time of treatment. It is suggested that equal cell densities should be used for measuring survival values and transformation induction. The quantitative results obtained are discussed in the light of the known mutagenic mechanisms of the chemicals tested. We also characterized morphologically transformed foci induced by different chemicals with respect to some biological properties. Anchorage independence was determined by testing growth in soft agar, loss of contact inhibition was quantitated by measuring maximum cell densities and malignancy was tested by tumor induction in nude mice. Although no very close correlation between these parameters and morphology was observed, the most malignant clones are also the ones with the highest values in the other tests. Our data make one or few genetical targets for transformation induction likely. We therefore speculate that the diverse phenotypes obtained might be due to differential activation of one or very few transforming genes in these cells.     

Comparison of oxygen consumption rates in minimally transformed BALB/3T3 and virus-transformed 3T3B-SV40 cells

In the recent years, bioenergetics of tumor cells and particularly cell respiration have been attracting great attention because of the involvement of mitochondria in apoptosis and growing evidence of the possibility to diagnose and treat cancer by affecting the system of oxidative phosphorylation in mitochondria. In the present work, a comparative study of oxygen consumption in 3T3B-SV40 cells transformed with oncovirus SV40 and parental BALB/3T3 cells was conducted. Such fractions of oxygen consumption as “phosphorylating” respiration coupled to ATP synthesis, “free” respiration not coupled to ATP synthesis, and “reserve” or hidden respiration observed in the presence of protonophore were determined. Maximal respiration was shown to be only slightly decreased in 3T3B-SV40 cells as compared to BALB/3T3. However, in the case of certain fractions of cellular respiration, the changes were significant. “Phosphorylating” respiration was found to be reduced to 54% and “reserve” respiration, on the contrary, increased up to 160% in virus-transformed 3T3B-SV40 cells. The low rate of “phosphorylating” respiration and high “reserve” respiration indicate that under normal incubation conditions the larger part of mitochondrial respiratory chains of the virus-transformed cells is in the resting state (i.e. there is no electron transfer to oxygen). The high “reserve” respiration is suggested to play an important role in preventing apoptosis of 3T3B-SV40 cells.     

Induction of ouabain-resistance mutation and cycle-dependent transformation of C3H/10T1/2 cells by N-nitroso-2-acetylaminofluorene

Ouabain-resistance mutation and cell cycle-dependent transformation were studied concurrently in the C3H/10T1/2 cell line treated with N-nitroso-2-acetylaminofluorene (N-NO-AAF) or N-nitroso-N-2-fluorenylacetamide. N-NO-AAF is a new direct-acting mutagen that exhibits a very short half-life (34 min) in complete medium independent of cell number seeded. With 0.1-0.3 mM of N-NO-AAF, cytotoxicity was noted after exposure for 2 h, but another phase of cytotoxicity was observed between 8 and 24 h. N-NO-AAF was more toxic than its parent compound 2-AAF. Moreover, maximal mutation frequency at the Na+/K(+)-ATPase gene locus (ouar mutation) was attained within 30 or 40 min of exposure, dependent on dosage of N-NO-AAF. With 2-AAF, 2-AF and 2-nitrofluorene, however, no detectable mutants were found under the same conditions. In cell cycle-dependent transformation assays, cells were synchronized by release from confluence-induced arrest of proliferation, 2 concentrations of N-NO-AAF were added for 2 h at various intervals during the cell cycle. The results clearly revealed that cells in 2 specific time intervals were susceptible to malignant transformation, i.e., at 10 and 18 h (early S phase) after release from the block.     

Mobile phone base station radiation does not affect neoplastic transformation in BALB/3T3 cells

A large-scale in vitro study focusing on low-level radiofrequency (RF) fields from mobile radio base stations employing the International Mobile Telecommunication 2000 (IMT-2000) cellular system was conducted to test the hypothesis that modulated RF fields affect malignant transformation or other cellular stress responses. Our group previously reported that DNA strand breaks were not induced in human cells exposed to 2.1425 GHz Wideband Code Division Multiple Access (W-CDMA) radiation up to 800 mW/kg from mobile radio base stations employing the IMT-2000 cellular system. In the current study, BALB/3T3 cells were continuously exposed to 2.1425 GHz W-CDMA RF fields at specific absorption rates (SARs) of 80 and 800 mW/kg for 6 weeks and malignant cell transformation was assessed. In addition, 3-methylcholanthrene (MCA)-treated cells were exposed to RF fields in a similar fashion, to assess for effects on tumor promotion. Finally, the effect of RF fields on tumor co-promotion was assessed in BALB/3T3 cells initiated with MCA and co-exposed to 12-O-tetradecanoylphorbol-13-acetate (TPA). At the end of the incubation period, transformation dishes were fixed, stained with Giemsa, and scored for morphologically transformed foci. No significant differences in transformation frequency were observed between the test groups exposed to RF signals and the sham-exposed negative controls in the non-, MCA-, or MCA plus TPA-treated cells. Our studies found no evidence to support the hypothesis that RF fields may affect malignant transformation. Our results suggest that exposure to low-level RF radiation of up to 800 mW/kg does not induce cell transformation, which causes tumor formation.     

Neutron-energy-dependent oncogenic transformation of C3H 10T1/2 mouse cells

The relative biological effectiveness (RBE) of a range of neutron energies relative to 250-kVp X rays has been determined for oncogenic transformation and cell survival in the mouse C3H 10T 1/2 cell line. Monoenergetic neutrons at 0.23, 0.35, 0.45, 0.70, 0.96, 1.96, 5.90, and 13.7 MeV were generated at the Radiological Research Accelerator Facility of the Radiological Research Laboratories, Columbia University, and were used to irradiate asynchronous cells at low absorbed doses from 0.05 to 1.47 Gy. X irradiations covered the range 0.5 to 8 Gy. Over the more than 2-year period of this study, the 31 experiments provided comprehensive information, indicating minimal variability in control material, assuring the validity of comparisons over time. For both survival and transformation, a curvilinear dose response for X rays was contrasted with linear or nearly linear dose responses for the various neutron energies. RBE increased as dose decreased for both end points. Maximal RBE values for transformation ranged from 13 for cells exposed to 5.9-MeV neutrons to 35 for 0.35-MeV neutrons. This study clearly shows that over the range of neutron energies typically seen by nuclear power plant workers and individuals exposed to the atomic bombs in Japan, a wide range of RBE values needs to be considered when evaluating the neutron component of the effective dose. These results are in concordance with the recent proposals in ICRU 40 both to change upward and to vary the quality factor for neutron irradiations.     

Fibroblast growth regulatory factor inhibits DNA synthesis in BALB/c 3T3 cells by arresting in G1

A cell surface macromolecular component from quiescent BALB/c 3T3 mouse cells (designated fibroblast growth regulatory factor, FGRF) inhibits DNA synthesis and cell division in growing 3T3 cells. Addition of FGRF to synchronized populations of growing 3T3 cells in the late G1 or early S phase did not inhibit DNA synthesis in the immediate S phase. However, a significant inhibition was observed in the S phase of the next round of cell cycle. Cells exposed to the regulatory factor in late S/early G2 or early G1 showed reduced DNA synthesis in the upcoming S phase; the late S/early G2 cells were more sensitive to inhibition than the cells in the G1. Further, the regulatory factor delayed the progression of G0/G1-arrested cells into the next S phase. These results suggest that the physiological effect of FGRF is to arrest cells in early G1, thus preventing their entry into a new round of cell cycle. In contrast to untransformed 3T3 cells, mouse cells transformed by SV40 were not subjected to growth-arrest by the regulatory factor, although the transformed cells contain active FGRF that inhibits DNA synthesis in growing 3T3 cells.     

Idiopathic myelofibrosis serum induces increased DNA synthesis in BALB/c 3T3 cells.

The effect of heated serum at a concentration of 10% in culture on the in vitro growth of confluent Balb/c 3T3 cells was studied in nine patients with Idiopathic Myelofibrosis and ten normal subjects. Patients showed significant increase in the mitogenic activity in comparison with normals. The growth factors conceivably implied for the observed effect are discussed. Particular attention is paid to Platelet-Derived Growth Factor from which serum mitogenic activity is primarily derived and is thought to take part in the genesis of bone marrow fibrosis.     

Overexpression of vinculin suppresses cell motility in BALB/c 3T3 cells.

The content of vinculin, a cytoplasmic protein found in focal contacts and cell-cell junctions, was increased in BALB/c 3T3 cells by gene transfection. The vinculin expressed from the full length chicken cDNA, incorporated into focal contacts and its pattern was identical to that of the endogenous protein. Cells stably expressing vinculin by 20% over the endogenous level had altered locomotory properties. In these cells, the ability to migrate into a wound formed in a confluent monolayer and the locomotion of individual cells were drastically reduced. The results provide direct evidence that cell locomotion can be regulated by modulating vinculin expression.     

Centriole ciliation and cell cycle variability during G1 phase of BALB/c 3T3 cells

Although variability in the duration of the cell cycle is thought to reflect growth-regulatory processes that control cell cycle progression, the precise timing of the variable period within the G1 phase of the cell cycle has not been defined. In particular, the timing of cell cycle variability in relation to the cell's commitment (R point) to the initiation of DNA synthesis remains controversial. In order to investigate cell cycle variability, indirect immunofluorescence was used to measure the formation of the primary cilium as a possible marker of G1 events in both stimulated quiescent and exponentially growing cells. The primary cilium, an internal "9 + 0" nonmotile structure formed by one of the interphase centrioles, was first detected in postmitotic BALB/c 3T3 cells 5 hr before the initiation of DNA synthesis, an interval similar to that for the reassembly of the primary cilium in serum-stimulated quiescent fibroblasts. This similarity in the timing of ciliation suggests that serum-stimulated quiescent cells reenter the cell cycle in early G1 and recapitulate much of G1. Moreover, the rate of cilia formation in both postmitotic and serum-stimulated quiescent cells was identical to the rate of DNA synthesis initiation. Thus, cell cycle variability occurs before ciliation in both stimulated quiescent and exponentially growing cells. Furthermore, since ciliation also precedes the R point, variability in the centriole cycle occurs before the R point and thus may reflect processes controlling the cell's commitment to the initiation of DNA synthesis.     

Photo catalogue for the classification of foci in the BALB/c 3T3 cell transformation assay

This catalogue is a display of focus photos representative of the BALB/c 3T3 cell transformation assay (CTA). It is intended as a visual aid for the identification and the scoring of foci in the conduct of the assay. A proper training from experienced personnel together with the protocol reported in this issue and the present photo catalogue will support method transfer and consistency in the assay results.