Of all the components of the culture medium, only CaCl2 induces DNA replication when added to resting cultures of Balb/c 3T3 cells. The effect is present even in a serum-free medium. Increasing the Ca++ concentration above the standard 1.8 mM in the medium of a new culture increases the total number of cells ultimately produced, without affecting the initial cell growth rate. This effect is synergistic with that of serum. The elevated Ca++ concentration also induces striking morphological changes. The Ca++ effect could not be reproduced by a Ca++ ionophore.These observations afford a new tool for studying how the various intracellular events following the addition of growth factors to resting cultures are involved in the control of cellular growth.In resting cultures of untransformed fibroblasts growth is stopped because medium components have become limiting (1-6). The limiting component is usually serum (7,8). In turn the limitation of the growth factors present in serum causes a series of changes of cellular properties, affecting the rates of transport of medium components to the cells, the intracellular concentration of cyclic nucleotides, and the rates of protein, RNA, and DNA synthesis. Addition of serum to a resting culture causes immediate changes in transport rates and cyclic nucleotide concentrations, followed by enhancement of the rates of protein and RNA synthesis, and ultimately by a resumption of DNA replication (9). It has been suggested that the various changes in the response to serum are coordinate effects (10); however, since serum contains many different growth factors, and hormones, the various observable effects could be brought about by separate mechanisms. A purified growth factor (FGF) (11) has been shown to cause several of those events, the most striking being an immediate and marked rise in cyclic GMP concentration (12, 13). Hence the question arises whether cGMP is the intracellular mediator of the growth-promoting activity of this and perhaps other growth factors.Examining the response of resting cultures of certain fibroblastic cells to the addition of medium components, we have obtained a striking activation of DNA synthesis by increasing the concentration of Ca++. This observation may further clarify the mechanism of growth activation. Since this effect does not require the presence of any macromolecular component in the medium, we may be a step closer to the intracellular mediator of the events responsible for the initiation of cell growth. MATERIALS AND METHODS Cultures

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The effects of phosphonoacetic acid on cell growth, expression of Epstein-Barr virus antigens, and virus production in human and marmoset lymphoblastoid cell lines have been studied. The drug had no significant effect at concentrations up to 100 jg/ml on cell growth or total cell DNA synthesis. Hi' er doses induced not only a drastic decrease in DNA synthesis and cell growth, but also a dramatic cell enlargement. Immunofluorescence studies showed that >30 jsg/ml of phosphonoacetic acid inhibited viral capsid antigen synthesis without affecting the expression of the nuclear antigen or the spontaneous and 5-iodo-2'-deoxyuridineinduced early antigens. Production of transforming EpsteinBarr virus was also blocked. Inhibition of viral capsid antigen expression and of virus production at low doses was reversible. The (human fibroblast) cells (3). PAA inhibits herpes simplex virus replication by interfering with the DNA polymerase activity (4). It has also been found to inhibit the replication of several other herpes viruses, including cytomegalovirus (5), Marek's disease virus (6), and equine abortus virus (7), and a non-herpes virus, vaccinia virus (8). In this paper we present data which show that PAA inhibits the synthesis of Epstein-Barr (EBV) viral capsid antigens (VCA) and of virus itself at concentrations which have no detectable effect on the synthesis of nuclear antigen (EBNA) and early antigens (EA), or on cellular DNA synthesis and cell proliferation. PAA also has no detectable effect on 5-iodo-2'-deoxyuridine-(IdUrd) or superinfection-induced EA synthesis in the nonproducer Raji cell line. MATERIALS AND METHODSCell Cultures. Lymphoblastoid cell lines B95-8, P3HR-1, nd Raji used in this investigation were obtained from Dr. 7eorge Klein and from Pfizer, Inc. The B95-8 cell line is an EBV-transformed marmoset lymphocyte line which proluces a lymphocyte-transforming virus (9). P3HR-1 and laji are Burkitt's lymphoma-derived lines which produce iontransforming cytopathic EBV (10) and no virus (11), respectively. Cells were routinely maintained in RPMI-1640 medium supplemented with 100 Ag/ml of streptomycin, 100 units/ml of penicillin, 300 jg/ml of glutamine, and fetal calf serum. They were fed every 3-4 days and the concentration was adjusted to 2 to 5 X 105 cells per ml at each feeding. All cultures were kept at 370 in a humidified 5% CO2 incubator.Reagents Cell Growth and DNA Synthesis. For measurement of DNA synthesis, B95-8 cells were seeded in duplicate at 5 X 105 per ml in a total of 3 ml for 48 hr and pulse labeled with 4 .Ci/ml of[3H]dThd (New England Nuclear, specific activity: 13 Ci/mmol) for 1 hr, and radioactivity ip the trichloroacetic-acid precipitable material was assayed in a Beckman liquid scintillation counter. For measurement of growth, cells were seeded in duplicate at 5 X 105 per ml and cultivated for various times. Viable cells were determined by the trypan blue exclusion technique. The effect of PAA on chromosomes was studied in exponentially growing B95-8 cells treated with co...

The ability of a mouse mammary tumor cell line to abrogate antibody neutralization of vesicular stomatitis virus was shown to be due to the presence of mycoplasma. The mycoplasma was isolated from the cell line and typed as Mycoplasma orale. Colonies of this mycoplasma were used to deliberately infect cell cultures which then gained the capacity to reactivate antibody-neutralized virus. The extent of the reactivation depended on the source of neutralizing antiserum. Other species of mycoplasma were tested and were found to reactivate neutralized virus, indicating that this may be a general phenomenon of mycoplasma contamination.