C3H2K Cells Research Articles

The effects of new cytochalasins from Phomopsis sp. and the derivatives on cellular structure and actin polymerization.

The effects of ten 10-phenyl-[11]cytochalasins produced by Phomopsis sp. including novel compounds having 5,7- or 6,7-glycol structures and their derivatives, on the cell morphology and actin distribution in C3H-2K cells, as well as on lymphocyte capping and actin polymerization, were examined. The structure-activity relationship reported in the previous papers has been confirmed. The novel glycol type compounds showed little or no activity, suggesting the importance of the perhydroisoindol-1-one nucleus for the manifestation of the cytochalasin actions.

Reversible induction of actin rods in mouse C3H-2K cells by incubation in salt buffers and by treatment with non-ionic detergents

Incubating conditions which induced actin paracrystal-like intracellular structures (actin rods) were investigated by using several cell lines. We have found that an incubation of cells of a mouse fibroblastic cell line, C3H-2K, in an isotonic solution of NaCl containing 1 mM MgCl 2, 1 mM CaCl 2 and 10 mM MES, pH 6.5, induced disintegration of stress fibers and formation of actin rods in the cytoplasm. Actin rods were induced also by incubating in salt buffers in which Na + of the above solution was substituted by most cations except K + or Rb +. When the actin rod-forming cells were transferred back to DMEM containing 10% FBS, actin rods disappeared and stress fibers subsequently re-formed within 1 h at 37 °C. Although the induction was observed in NaCl buffer at a wide range of pH values (5.5–10), the optimal pH was 6.5. Formation of actin rods is dependent upon cellular metabolism, as it was inhibited at 4 °C, or by metabolic inhibitors. Incubation in NaCl buffer induced actin rods in HeLa, L, NRK, BALB/c 3T3 and Swiss 3T3 cells, but not in CEF or MEF cells. A decrease in cell volume was observed parallel with the induction of actin rods, except for CEF and MEF cells. Alterations in intracellular concentrations of Na, K or Ca were not correlated with the induction, however. Actin rods were also induced in C3H-2K cells by a brief treatment with non-ionic detergents. Tween 80 at concentrations as low as 0.003% was effective for the induction, but did not increase the passive membrane transport of p-nitrophenylphosphate. In contrast to the induction by NaCl buffer, treatment with Tween 80 induced numerous tiny actin rods at 4 °C, which became larger when further incubated at 37 °C. Double immunofluorescence staining with anti-actin antibody and anti-vinculin antibody showed that vinculin plaques remained at least in an early stage of the actin rod formation. We discuss the mechanism for the induction of actin rods based upon the present findings.

Structure-activity relationship of thirty-nine cytochalasans observed in the effects on cellular structures and cellular events and on actin polymerization in vitro.

The effects of twenty-three natural cytochalasans and sixteen synthetic derivatives on actin-distribution and alteration in morphology of C3H-2K cells, inhibition of lymphocyte capping, and inhibition of actin filament elongation were compared. The effects on cellular level and the in vitro effects showed positive correlation and the structure-activity relationship observed is discussed.

Demonstration of a proliferation regulatory factor (PRF) in normal human urine, and its purification.

A material(s) affecting the proliferation of eukaryotic cells, such as C3H2K cells, and prokaryotic cells, such as E. coli, was found in the urine of normal healthy men and was concentrated in a certain fraction, "lysin-Sepharose eluate," of the urine. The material was named proliferation regulatory factor, PRF. With increase in the concentration of PRF, the proliferation rate of normal eukaryotes and prokaryotes increased to maxima of no more than twice those of untreated cells. On further increase in the concentration of PRF the growth rates of these cells decreased to the rates of untreated cells. PRF enhanced the rates of synthesis of DNA and RNA in C3H2K cells about 2-fold, but strongly suppressed the rates of growth, DNA synthesis, and RNA synthesis of cancerous cells, such as HeLa and MH134/M cells. PRF was purified from the "lysine-Sepharose eluate" by DEAE-cellulose column chromatography and gel filtration on a Sephadex G-100 column. PRF consisted of three molecules with molecular weights of 41,000, 7,800, and 5,100, which were named PRF-I, II, and III, respectively. The lysine-Sepharose eluate and purified PRF's had similar effects on the proliferation of eukaryotes and prokaryotes.

A new tumor promoter, dihydroteleocidin B, enhances cell growth and the production of murine leukemia virus by fibroblasts.

The effects of tumor promoters on the expression of murine leukemia virus (MuLV) were studied in tissue culture. Dihydroteleocidin B, an indole alkaloid, recently found to be a tumor promoter, enhanced not only the production of Moloney MuLV (M-MuLV) by a mouse fibroblast cell line, C3H2K, persistently infected with M-MuLV, but also growth on the C3H2K cells. The production of infectious M-MuLV by M-MuLV-infected C3H2K cells that had been treated with dihydroteleocidin B for 1-7 days was four or five times higher than that of control cells. C3H2K cells grew faster and became stationary at higher cell densities in the presence of dihydroteleocidin B than in its absence. The tumor-promoting phorbol ester phorbol-12, 13-didecanoate and 12-O-tetradecanoylphorbol-13-acetate also enhanced the production of M-MuLV, but their effects were not so strong as that of dihydroteleocidin B. These tumor promoters, however, did not induce production of endogenous MuLV in C3H2K or K-BALB cells.

Further characterization of a thermosensitive transformation variant of mouse fibroblasts.

Temperature-sensitive (ts) variants that express phenotype at low (33 degrees) but not at high (38.5 degrees) temperature were isolated from a mouse fibroblast strain C3H2K cells. Among these variants, cloned ts-12B cells showed at 38.5 degrees a density-dependent inhibition of growth typical of normal fibroblasts cultured in vitro. However, at low temperature they lost this capacity and grew to a higher saturation density. The ts variant was also temperature-sensitive as regards serum requirement: it required a higher concentration of serum for growth at 38.5 degrees than at 33 degrees. However, the cells behaved at both temperatures like the parent strain, possessing anchorage-dependence for growth and fibronectin, whereas they were like transformed cells with respect to release of high fibrinolytic activity. No type-C virus core protein p30 was detected at either temperature. Thus, various parameters of transformation in vitro were independently regulated in these variant cells.

Studies on Polyoma Virus DNA Replication in Synchronized C3H2K Cells

In G1-arrested cells infected between 1 and 12 h after having been stimulated by fresh serum to progress to S phase, polyoma virus DNA synthesis proceeded in the first half of S phase, and virus and whole cellular DNA accumulated at about the same time. However, in cells infected later than 14 h after serum stimulation, virus DNA synthesis was shifted to the next S phase. Thus, a permissive cell attains competence for polyoma virus DNA replication at a precise moment during an S phase initiated by fresh serum, which can efficiently replace the early virus host DNA stimulation function. When cells were incubated in serum that had lost its capacity to stimulate host DNA synthesis by pre-absorption with growing cells, normal yields of polyoma DNA could nevertheless be observed, which shows that extensive replication of host DNA does not seem to be an obligatory condition for virus DNA replication.

Regulation of the Induction of Cellular DNA Synthesis in a Mouse Cell Clone by Bovine Adenovirus Type 3: Effect of Heat Treatment

Bovine adenovirus type 3 (BAV3) causes abortive infection and induces cellular DNA synthesis in the mouse cell line, C3H2K.A clone, N-1, was selected from this cell line after infection with BAV3 in the presence of 5-fluorodeoxyuridine. In this clone, cellular DNA synthesis was not induced by BAV3 infection, but became inducible in N-1 cells when the temperature of the culture was raised to 41°C-42°C prior to infection by BAV3. Heat-activated N-1 cells again became insensitive to BAV3 infection after prolonged cultivation at the normal temperature of 37°C. The addition of cycloheximide did not prevent activation of N-1 cells by heat, but it did prevent the activated N-1 cells from returning to their original state of being insensitive to BAV3 infection when cultivated at 37°C. Viral mRNA synthesis, which was followed by the induction of cellular DNA synthesis, was transiently detected only when N-1 cells were heat-treated before virus infection. Penetration into the cell and the uncoating of BAV3 proceeded in N-1 cells almost as normally as in C3H2K cells. Though the N-1 clone was selected by a procedure involving BAV3 infection, N-1 cells did not contain a detectable amount of the BAV3 DNA sequence as determined by analysis of DNA-DNA reassociation kinetics. The significance of these results is discussed.

Propagation of a B-Tropic Murine Leukemia Virus in N-Type Cells in the Presence of Different Murine Sarcoma Viruses

B-tropic MuLV, WN1802B, was converted to NB-tropic after 5 days of propagation in its nonpermissive N-type MLg or C3H2K cells in the presence of the S + L - MuSV, but not in the presence of the Harvey or Kirsten strains of MsSV.

Enhancement of SV40 transformation by treatment of C3H2K cells with uv light and caffeine

Abstract The transformation frequency of C3H2K cells infected with SV40 was enhanced by treatment of cells with uv light and/or caffeine. This enhancement depended upon: (a) multiplicity of infection (m.o.i.) of the virus; (b) cell phase at time of infection; and (c) length of exposure to caffeine. The transformation frequency of C3H2K cells was proportional to the viral dose between an m.o.i. of 103 and 104 (one-hit response). Treatment of cells with uv (75 ergs/mm2) or caffeine (1 mM) enhanced the transformation frequency without affecting the linearity of the reaction. When irradiated cells were infected with virus and incubated with caffeine, the transformation frequency was not proportional to the virus dose, and far exceeded the expected response if uv and caffeine act additively. The maximum transformation frequency obtained was 45%. Experiments with partially synchronized cultures indicated that cells passing through the late S period was highly susceptible to the transformation. However, the enhancement of transformation by uv and/or caffeine did not appear to depend upon a particular period in the cell cycle. The transformation frequency of unirradiated cells increased progressively during a 4-day incubation with 1 mM caffeine, after which time it leveled off. In contrast, when cells were uv-irradiated prior to virus infection and then incubated in the presence of caffeine, the enhancement of transformation appeared in the early period of incubation, reaching a maximum within 24 hr. The latter time course was coincident with that of the repair process of uv-damaged DNA, as judged from unscheduled DNA synthesis.

Enhancement of SV40 transformation by treatment of C3H2K cells with UV light and caffeine: I. Combined effect of UV light and caffeine

Treatment of cultured mouse cells, C3H2K, with UV light and/or caffeine enhanced the frequency of SV40-induced transformation. This enhancement depends upon the doses of UV and caffeine and the mode of combination of these agents. Irradiation of cells with increasing doses of UV just before infection resulted in approximately 2-fold enhancement of the transformation frequency up to a dose of 90 ergs/mm 2 and 3.3-fold at 150 ergs/mm 2. Addition of 1 m M caffeine to the medium for 4 days subsequent to infection brought about a 2-fold enhancement. When cells were irradiated and treated with 1 m M caffeine, the enhancement was approximately 4-fold up to a UV dose of 90 ergs/mm 2 and 5.9-fold at 150 ergs/mm 2. When 0.1–4 m M caffeine was added for 4 days postinfection, the absolute number of transformations increased, and an enhancement ratio of 1.3–6.8 resulted. After the addition of the same increasing doses of caffeine to UV-irradiated cells (75 ergs/mm 2), the enhancement of transformation frequency was even higher ranging 2.0 to 13.3. The transformation frequencies thus obtained by the double treatment were always higher than those predicted if UV and caffeine acted additively. The transformation frequency was little affected by the addition of dibutyrylcyclic AMP and theophylline.

Discrimination of carcinogenic activity and inactivation of transforming activity of hydrocarbons, by .BETA.-lactoglobulin.

Comparative study was made on association of the carcinogenic and the non-carcinogenic hydrocarbons with β-lactoglobulin. It was concluded that β-lactoglobulin preferentially associated with the carcinogenic hydrocarbons. The capacities of 1g of β-lactoglobulin to associate with 3, 4-benzpyrene and 9, 10-dimethyl 1, 2-benzanthracene, virulent carcinogens, were 63.0 and 32.8mg respectively, while those with 1, 2-benzpyrene and chrysene, limited-and non-carcinogenic analogues, were 8.2 and 0.5mg respectively. Casein and soybean protein failed to display the preferential association with the carcinogens. As a result of complex formation with β-lactoglobulin, the toxicity of 3, 4-benzpyrene against the cultured cell line, C3H-2K, was neutralized to 1/10 and the transforming activity of the carcinogen against C3H-2K cells was significantly lowered.

Rapid isolation of nucleoli from detergent purified nuclei of various tumor and tissue culture cells

A rapid isolation procedure of nucleoli from detergent purified nuclei of some tumor and tissue culture cells is described. The procedure makes use of a non-ionic detergent, Nonidet P40 and sodium deoxycholate to purify nuclei followed by the addition of Ca 2+ or Mg 2+ to strengthen the nucleoli against sonication. Enzymatically active (with respect to nucleolar RNA polymerase) nucleoli containing undegraded nucleolar RNAs may be isolated from a mouse hepatoma MH134, Ehrlich ascites tumor, HeLa cells, L cells and C3H2K cells with this procedure.

Effect of arabinosyl-cytosine on the G 1-arrested cells which were subsequently induced to divide by medium change

Treatment of C3H2K cells arrested in the G 1 phase with arabinosyl-cytosine (araC) for 2 h caused an unexpected delay of DNA synthesis when the cells were subsequently induced to divide by medium change. The araC-induced delay was reversed by excess amount of deoxycytidine. The higher the concentration of araC, the longer the delay period. The delay period was invariable whether the cells were treated with araC from 0 to 2 h or from 6 to 8 h, i.e., early or late G 1. Similar delay was produced by ethidium bromide and fluorodeoxyuridine.

Transient inhibition of mitotic activity by addition of calf serum

A transient depression of mitotic activity occurred immediately after addition of fresh calf serum to the cultures of C3H2K cells; the extent of the depression depended upon the serum concentration.

Transformation of a mouse cell line by murine sarcoma virus (Moloney).

AbstractMurine sarcoma virus (Moloney) (MSV) transformed C3H2K cells originating from the kidney tissues of a newborn C3H/He mouse. Titration of the virus showed a one‐hit dose response in the presence of an excess of Friend leukemia virus (FLV) and a two‐hit dose response in the absence of FLV, indicating that MSV was defective and a dual infection by MSV and a murine leukemia virus was required for MSV focus formation. Preinfection with FLV interfered with focus formation by MSV. The MSV foci appeared most abundantly when the cells in the S phase were infected with the virus. Remarkable similarities between murine and avian sarcoma viruses were thus established. Focus center assay on either uninfected or FLV‐infected C3H2K cell monolayer indicated that MSV foci did not arise by cellular division alone but required secondary infection, while RSV foci arose by cellular division alone.

Induction of cell replication

C3H2K cells in the stationary phase divided synchronously after renewal of the culture medium. The maximum DNA synthesis was at about 20 h after medium change and mitosis 10 h later than DNA synthesis. At the peak of the DNA synthesis, approx. 50 per cent of the cells were in S phase. There were, at least, two peaks of RNA synthesis, just after the medium change and before the DNA synthesis. The factor inducing the cell division was contained in calf serum. The contact with fresh medium for 2.5 h induced the division of 50 per cent of the cells. The factor inducing the cell division was rapidly consumed in contact with the cell monolayer: it was lost almost completely within 24 h.

Requirement of cellular DNA synthesis for the growth of Friend leukemia virus

Cytosine arabinoside, a specific inhibitor of DNA synthesis, inhibited the growth of Friend leukemia virus (FLV) when added in the early phases of infection. Infecting the synchronized cultures of C3H2K cells, the author found that cellular DNA synthesis was required for the growth of FLV in the early phases of infection.