On the mechanism of elevated prostaglandin E2 production in 3T3 fibroblasts transformed by polyoma virus

Polyoma-virus-transformed 3T3 fibroblasts (py 3T3 cells) produce considerably more prostaglandin E2 than regular 3T3 cells during growth in cell culture. Incubations with exogenous arachidonic acid showed no increase in prostaglandin-producing capacity in the transformed cells. The rates of degradation of prostaglandin E2 were similar in the two lines. After labeling of cells with [1-14C]arachidonic acid, py 3T3 cultures continuously released radioactivity while the release by regular 3T3 cells was almost completed after 3 h. Prostaglandin E2 production during short incubations in buffer at various times after medium change was constantly higher in the transformed cells. Furthermore, hydrocortisone completely inhibited prostaglandin synthesis by the transformed cells. These results suggest that the increased formation of prostaglandin by py 3T3 cells is due to continuously elevated activity of phospholipase A2 or another acyl hydrolase.     

Effect of exogenous fatty acids on the retention of phospholipid acyl groups by mouse L fibroblasts

Exogenous oleic or linoleic acid, given at a high but nontoxic level (1 mg fatty acid/day for 20 . 10(6) cells in 50 ml medium), caused substantial redistribution of the otherwise permanently retained phospholipid acyls in mouse L fibroblasts. 18--40% of the preformed phospholipid acyls were shifted to triglycerides but most returned to phospholipids when the supply of exogenous fatty acid was removed. The phospholipid acyls could be reshuttled back to triglycerides again whenever an adequate amount of exogenous fatty acid was provided. Daily changes of medium containing oleic acid bound to bovine serum albumin caused a still greater total loss of phospholipid acyls into the medium. The removal of the prelabeled phospholipid acyls also occurred with phospholipid acyls which had been synthesized from [1-(14C)]acetate 3 days earlier. The results demonstrate the fact that the apparent permanently retained phospholipid acyl groups found in L-cells could in fact be displaced through experimental manipulations.     

Elo1p-dependent carboxy-terminal elongation of C14:1Delta(9) to C16:1Delta(11) fatty acids in Saccharomyces cerevisiae

Saccharomyces cerevisiae medium-chain acyl elongase (ELO1) mutants have previously been isolated in screens for fatty acid synthetase (FAS) mutants that fail to grow on myristic acid (C14:0)-supplemented media. Here we report that wild-type cells cultivated in myristoleic acid (C14:1Delta(9))-supplemented media synthesized a novel unsaturated fatty acid that was identified as C16:1Delta(11) fatty acid by gas chromatography-mass spectroscopy. Synthesis of C16:1Delta(11) was dependent on a functional ELO1 gene, indicating that Elo1p catalyzes carboxy-terminal elongation of unsaturated fatty acids (alpha-elongation). In wild-type cells, the C16:1Delta(11) elongation product accounted for approximately 12% of the total fatty acids. This increased to 18% in cells that lacked a functional acyl chain desaturase (ole1Delta mutants) and hence were fully dependent on uptake and elongation of C14:1. The observation that ole1Delta mutant cells grew almost like wild type on medium supplemented with C14:1 indicated that uptake and elongation of unsaturated fatty acids were efficient. Interestingly, wild-type cells supplemented with either C14:1 or C16:1 fatty acids displayed dramatic alterations in their phospholipid composition, suggesting that the availability of acyl chains is a dominant determinant of the phospholipid class composition of cellular membranes. In particular, the relative content of the two major phospholipid classes, phosphatidylethanolamine and phosphatidylcholine, was strongly dependent on the chain length of the supplemented fatty acid. Moreover, analysis of the acyl chain composition of individual phospholipid classes in cells supplemented with C14:1 revealed that the relative degree of acyl chain saturation characteristic for each phospholipid class appeared to be conserved, despite the gross alteration in the cellular acyl chain pool. Comparison of the distribution of fatty acids that were taken up and elongated (C16:1Delta(11)) to those that were endogenously synthesized by fatty acid synthetase and then desaturated by Ole1p (C16:1Delta(9)) in individual phospholipid classes finally suggested the presence of two different pools of diacylglycerol species. These results will be discussed in terms of biosynthesis of different phospholipid classes via either the de novo or the Kennedy pathway.     

LY-2+ effectors cytotoxic for syngeneic tumor cells: generation by allogeneic stimulation and by supernatants from mixed leukocyte cultures

The present study was aimed at gaining insight into means by which stimulation of mouse spleen cells with allogeneic normal cells in mixed leukocyte cultures (MLC) can result in the generation of effector cells cytotoxic for syngeneic tumor or transformed cells. Stimulation of lymphocytes from BALB/c or C3H mice for 5 days with cells from mice of every allogeneic strain tested, in medium containing mouse serum and lacking xenogeneic serum, resulted in the activation of effectors cytotoxic for syngeneic cells transformed spontaneously or by SV40, polyoma or adenovirus. In each experiment, all of the syngeneic transformed cell lines, as well as clones derived from these lines, were lysed to the highest degree by effectors obtained from the same culture, and therefore stimulated with cells from the same allogeneic strain. Although the particular allogeneic sensitizing strain that induced the highest cytolytic activity varied between experiments, effectors obtained from the culture with the highest cell recovery always exhibited the greatest cytotoxicity against all the syngeneic transformed cells and clones. Lysis was mediated predominantly by Ly-2+ effectors; total lytic units of cytotoxicity recovered after treatment with monoclonal anti-Ly-2 antibody and complement (C) were reduced by 85 to 90% compared to cells treated with C alone. Lysis of syngeneic tumor cells by the allosensitized effectors in cytotoxicity assays was not inhibited by the addition of unlabeled "blocking" lymphocytes from the allogeneic strain used for sensitization. In addition, it was found that lymphocytes cultured without stimulating cells for 5 days in medium supplemented with supernatants from secondary MLC that are known to contain high levels of lymphokines, mediated high levels of cytotoxicity on all the transformed cells tested, but lacked detectable cytotoxic activity for syngeneic or allogeneic Con A blasts. The MLC supernatant-activated effectors that lyse the transformed cells are phenotypically CTL, because treatment with anti-Ly-2 and C reduced lytic activity by approximately 75%. Taken together, these findings suggest that the generation in MLC of Ly-2+ effector cells cytotoxic for syngeneic transformed cell lines might not be due, in some cases, to lymphocyte responses to particular alloantigens on the stimulating cells that are cross-reactive with "alien" histocompatibility antigens on transformed cells, but rather is due to effector cell activation by lymphokines produced during allogeneic stimulation.     

Metabolism of cholesterol in normal hamster fibroblasts and those transformed by the SV 40 virus. Effect of low density lipoproteins

The amount of cholesterol and the percentage of esterified cholesterol were increased in transformed cells. The cholesterol synthesis from [14C] sodium acetate was reduced and cholesteryl oleate uptake increased by 3 fold in transformed cells. The activity of acyl coenzyme A-cholesterol-acyltransferase, measured in situ was also increased in transformed cells. Studies with 125I-LDL pointed out an increase of binding, and especially of internalization of LDL by transformed cells. Finally, long term culture in a lipoprotein-deficient medium showed that transformed cells exhibited a higher ability (tested by growth rate and cholesterol synthesis) to adapt themselves to lipid depletion.     

Ammonia effects in cultures of normal and transformed 3T3 cells

3T3 and SV-40 transformed 3T3 mouse fibroblasts were cultured in media with serum and antibiotics plus ammonia (NH₃ z NH₄⁺) added as NH₄C1. Both cell lines cultured without added ammonia showed normal morphology and multiplication even though ammonia in the medium at the end of the culture period ranged from 35 to 48 μg/ml. Ammonia concentrations being significantly higher in media removed from cells at the end of the culture period than in media incubated identically without cells, verified that cells released substantial quantities of ammonia in addition to components of the medium which underwent spontaneous breakdown. Both cell lines showed changes in morphology and highly significant reductions in cell multiplication which increased progressively as the concentration of added ammonia on the initial day of culture was increased to 35μg/ml. Control 3T3 cultures released significantly greater quantities of ammonia per cell than control cultures of transformed cells but their multiplication was more adversely affected by added ammonia. There were downward shifts in pH of the culturing medium for both cell lines as culture age increased at all concentrations of added ammonia, However, significant reductions in cell multiplication resulted from additions of ammonia that did not produce significant changes in extracellular pH. The data show that studies upon the effects of pH of the medium on cultured cells require control of ammonia concentrations.     

Transforming viruses directly reduce the cellular growth requirement for a platelet derived growth factor

Early passage mouse embryo fibroblasts, mouse 3T3 cell lines, and early passage diploid human fibroblasts grew to higher cell densities in tissue culture medium supplemented with serum than in medium supplemented with defibrinogenated platelet-poor plasma (PPP). Unlike the mouse cells, the human fibroblasts displayed this differential growth response only in the presence of hypophysiologic concentrations of calcium. The addition of heat-treated extracts of human platelets to PPP-supplemented medium stimulated the replication of both the normal mouse cells and early passage human embryo fibroblasts. Human or mouse fibroblasts transformed by either retroviruses or by SV40, including SV40 infected “serum revertants” and “flat transformants,” grew to equal cell densities in medium supplemented with either serum or PPP. Infection of Balb/c-3T3 cells with SV40 rapidly induced them to grow in PPP-supplemented medium demonstrating that the ability of SV40-transformed cell lines to proliferate in PPP-supplemented medium does not arise from the cell culture selection procedures usually employed to obtain stable virus-transformed cell lines. 3T3 cells infected but not transformed by retroviruses do not replicate in PPP-supplemented medium demonstrating that reduction of the growth requirement for the platelet growth factor(s) by retroviruses is a transformation-specific response. Cell cultures that did not proliferate well in PPP-supplemented medium did not form tumors when inoculated into athymic nude mice. Many, although not all, of the lines which grew well in PPP medium were tumorigenic in nude mice. Together, these findings indicate that: (1) normal fibroblast-like cells display a growth requirement for factor(s) present in serum but not found in PPP; (2) this serum specific growth factor is derived from platelets; (3) a primary response to viral transforming genes is a reduction in the growth requirement for these platelet-derived factors; and (4) cells that have a reduced requirement for the platelet-derived growth factor are often tumorigenic.     

Release of phospholipid acyl groups from fetal human fibroblasts

Human diploid fibroblasts incorporate [1-¹⁴C]oleate primarily into phospholipids; this radioactivity is gradually lost by cells of adult origin, but retained, after an initial loss, by fetal cells during growth in unlabeled unchanged medium. [1-¹⁴C]oleate is released from fetal cells after each subsequent medium change even when conditioned medium is used. Unlabeled cells readily incorporate medium radioactivity previously released by labeled cells. Raising the serum concentration of the medium increases acyl group release. The apparent retention of acyl groups by fetal cells in the absence of medium changes results from a dynamic equilibrium of fatty acid moieties between cellular phospholipids and the culture medium.     

Analysis of a transgenic mouse containing simian virus 40 and v-myc sequences.

Two plasmids, one containing the simian virus 40 (SV40) genome and the mouse metallothionein I gene and one containing the v-myc gene of avian myelocytomatosis virus MC29, were coinjected into mouse embryos. Of the 13 surviving mice, one, designated M13, contained both myc and SV40 sequences. This mouse developed a cranial bulge identified as a choroid plexus papilloma at 13 weeks and was subsequently sacrificed; tissue samples were taken for further analysis. Primary cell lines derived from these tissues contained both myc and SV40 DNA. No v-myc mRNA could be detected, although SV40 mRNA was present in all of the cell lines tested. T antigen also was expressed in all of the cell lines analyzed. These data suggest that SV40 expression was involved in the abnormalities of mouse M13 and was responsible for the transformed phenotype of the primary cell lines. Primary cell lines from this mouse were atypical in that the population rapidly became progressively more transformed with time in culture based on the following criteria: morphology, growth rate, and the ability to grow in soft agar and in serum-free medium. The data also suggest that factors present in the mouse regulated the ability of SV40 to oncogenically transform most cells and that in vitro culture of cells allowed them to escape those factors.     

Circumvention of the methotrexate transport system by methotrexate-phosphatidylethanolamine derivatives: effect of fatty acid chain length

Methotrexate has been conjugated (amide bond) via either the alpha or gamma, or both alpha and gamma, glutamyl carboxyl groups to the amino function of dihexanoylphosphatidylethanolamine (C6C6PE) and 1-tetradecanoyl-2-hexanoylphosphatidylethanolamine (C14C6PE). These phospholipid prodrugs (either free or incorporated into liposomes) were compared with the corresponding ditetradecanoylphosphatidylethanolamine (C14C14PE) conjugates, some of whose properties have been described previously, for their ability to inhibit the proliferation of human leukemic cells (CEM/O) or cells derived therefrom (CEM/MTX) that are resistant to methotrexate because of a defective drug transport system. Regardless of chain length, the gamma conjugates were more effective than either the alpha or the alpha, gamma conjugates, in inhibiting growth of the parent cells, confirming initial experiments with mouse cells. Chain length had, however, a pronounced influence on the capacity of the various gamma derivatives to circumvent the transport defect. For example, CEM/MTX cells were 120-fold less susceptible than CEM/O cells to inhibition by either methotrexate or methotrexate-gamma-C6C6PE, whereas both cell lines were equally sensitive to methotrexate-gamma-C14C14PE. Although less potent than either of the foregoing, methotrexate-gamma-C14C6PE could partially by-pass the defective transport system. These results suggest that methotrexate-gamma-PE derivatives with appropriate acyl residues might be useful probes to investigate the mechanism by which phospholipids in general are able to traverse cell membranes.     

Biosynthetic labelling of membrane lipids of eukaryotic cells in tissue culture by a novel type of fluorescent fatty acids.

W-Anthryl labelled fatty acids with hydrocarbon chains of different lengths (C8, C11, C15) and different degrees of unsaturation have been incorporated into the membrane lipids of three different cell lines in tissue culture by addition of these 3H-labelled precursor fatty acids to the growth medium. The cell lines were baby hamster kidney cells (BHK 21), Chang liver cells and the RN6 cell line derived from a chemically induced Schwannoma tumor cell clone. Cell growth was normal. The quantitative analysis on the basis of radioactivity determinations demonstrated that the fluorescent-labelled fatty acids were introduced into the neutral lipid fraction (triglycerides, diglycerides, and cholesterol esters, all present in small amounts), but mainly into the phospholipid classes phosphatidylcholine, -ethanolamine and -serine, and to a lesser extent, as N-acyl component of sphingolipids (sphingomyelins, ceramides, mono- and diglycosylceramides). Cell fractionation studies indicated that the membranes of all subcellular particles were labelled with the fluorescent probes in their lipid moieties. These w-anthryl fatty acids are the first type of fluorescent lipid precursors which can be incorporated biosynthetically in vivo into membrane lipids of eukaryotic cells. The effective incorporation of the bulky fluorescent anthryl group in the terminal position of fatty acids of different chain lengths into the complex membrane lipids of the cell gives proff of 1) their uninhibited membrane transport, 2) their activation by the acyl-CoA synthetase and 3) their substrate properties for the O- acyl and N-acyl transferases in phospho- and sphingolipid biosynthesis.     

Serum-free growth of normal and transformed fibroblasts in milk: differential requirements for fibronectin

Bovine milk may be used as a supplement for the serum-free growth of certain fibroblastic cells in culture. The growth properties of three representative cell types in milk-supplemented medium were examined; fibroblastic cell strains, fibroblastic cell lines, and transformed fibroblasts. Transformed fibroblasts, which included RNA and DNA tumor virus-transformed cells and carcinogen-transformed cells, grew in milk. Instead of growing attached to the culture dishes, as they normally do in serum, transformed fibroblasts grew in milk as large clusters in suspension. In contrast, nontransformed fibroblastic cell strains and cell lines did not grow in milk-supplemented medium. Fibroblasts transformed by a temperature-sensitive transformation mutant of Rous sarcoma virus were temperature-sensitive for growth in milk. The failure of cells to adhere to the substratum in milk-supplemented medium suggested that milk might be deficient in attachment factors for fibroblasts. When the attachment of fibroblastic cells in milk- supplemented medium was facilitated by pretreating culture dishes with fibronectin, (a) transformed cells grew attached rather than in suspension, (b) normal cell lines attached and grew to confluence, and (c) normal cell strains adhered and survived but did not exhibit appreciable cell proliferation.     

向日葵Ti T—DNA转化系的原生质体培养和细胞培养

根癌农杆菌离体感染向日葵子叶、下胚轴外植体形成的Ti T-DNA转化组织在激素条件下长期继代培养后,用来进行原生质体培养和细胞培养。适于B6S3转化系和T37转化系原生质体培养的培养基分别为附加不同激素和糖类的C81V和DPD培养基。用液体浅层法培养3～5天时,原生质体开始分裂。10天后形成细胞团。B6S3转化系还可直接从原生质体产生原胚状结构。转化系的细胞克隆均保持着激素自主型生长特性和冠瘿碱合成酶合成特性。     

Immortalized mouse brain endothelial cells are ultrastructurally similar to endothelial cells and respond to astrocyte-conditioned medium

Summary Studies of brain microvessel endothelial cell physiology and blood-brain barrier properties are often hampered by the requirement of repeatedly producing and characterizing primary endothelial cell cultures. The use of viral oncogenes to produce several immortalized brain microvessel cell lines has been reported. The resulting cell lines express many properties of the blood-brain barrier phenotype but do not completely mimic primary endothelial cells in culture. As immortalized brain microvessel endothelial cell lines have not yet been produced from mice, we transformed mouse brain endothelial cells with the adenovirus E1A gene using a retroviral vector (DOL). Eight of 11 clones produced exhibited an endothelial-like cobblestone morphology and were characterized as endothelial with a panel of antibodies, lectins, and ultrastructural criteria. These cells are endothelial in origin and share ultrastructural features with primary cultures of endothelial cells. Examination of freeze fracture and transmission electron micrographs show adherens junctions exist between the transformed cells, and culture in astrocyte-conditioned medium induces the formation of gap junctions. This is one indication that responses to astrocyte-derived factors are retained by the transformed cell lines.     

A glycine-enriched astrocytic cell clone derived from mouse cerebella transformed in vitro by simian virus-40

Measurements were made of the amino acid content of a cellular clone (K55) derived from mouse cerebellar cultures transformed in vitro by simian virus-40 (Alliot and Pessac, 1981) and that appears to be astroglial. Both the total amount of amino acids as well as the percentage of glycine in K55 cells were higher than in the mixed cultures from which they are derived. Further, glycine accumulates in the culture medium of K55 cells, but not in the medium of the parental mixed cell culture (C14), thereby suggesting that glycine is synthesized and released by K55 cells.     

Induction of IL 2 responsiveness in a murine IL 3-dependent cell line

A mouse IL 3-dependent cell line, FD.C/1, can be induced to IL 2 growth responsiveness by culture in IL 2-conditioned culture medium. The IL 2-dependent cell lines derived by this procedure have been designated FD.C/2 cells. Once established, the FD.C/2 cells respond to human, rat, and mouse IL 2. When cultured with murine IL 3, FD.C/2 cells did not proliferate, appearing to accumulate in the G1 phase of the cell cycle. Other human and mouse lymphokines failed to stimulate FD.C/2 cell growth. The growth dependence of FD.C/2 cells on IL 2 could not be reversed to IL 3. Cell lines derived by these procedures could provide in vitro models for hemopoietic differentiative events.     

Alveolar pre-type II cells from the fetal rabbit lung: effect of confluence on the production of disaturated phosphatidylcholine

Pre-type II alveolar cells isolated from the fetal rabbit lung on the 24th gestational day have been maintained in vitro for 14 days in a chemically defined medium supplemented with hormone-stripped serum. These cells replicate in culture. Measurement of the incorporation of [14C]choline into cellular disaturated phospholipid indicated that those cells grown in vitro under standard conditions for 8 days (pre-confluent) incorporate the radioactive precursor at a similar rate to cells maintained for 14 days (post-confluent). Both dexamethasone and serum-free medium conditioned by monolayer cultures of fetal rabbit lung fibroblasts stimulated [14C]choline incorporation into disaturated phosphatidylcholine (PC) by the pre- and post-confluent cultures after 24 or 48 h of exposure: the conditioned medium was more effective than the steroid. These treatments had little effect on choline incorporation into disaturated phosphatidylcholine of preconfluent cells during the first 12 h. A marked response occurred by 24 h after which the labelling of disaturated phosphatidylcholine plateaued. In contrast, with post-confluent cells labelling of disaturated PC increased in a more linear fashion and only plateaued after 72 h. Determination of the ratio of incorporation of [14C]choline into disaturated versus unsaturated phospholipid indicated that serum-free medium conditioned by monolayer cultures of fetal lung fibroblasts specifically increased the level of radioactive precursor in the disaturated phospholipid in both the pre- and post-confluent cell monolayers.     

Increased saturated phospholipid in cultured cells grown with linoleic acid

Summary We found that fetal bovine serum supplementation of culture medium provided limited quantities of linoleic acid, an essential fatty acid, to cells grown in culture (2.8 ± 0.3% of total fatty acids in 12 lots). Supplementation of the medium with additional linoleic acid resulted in altered phospholipid acyl composition in cells of two established lines, A549, a putative model of the pulmonary Type II epithelial cell, and SIRC, a line derived from rabbit corneal epithelium. In particular, linoleic acid supplementation induced a relative increase in disaturated choline phosphoglycerides of 33 and 36%, respectively, in cells of the two lines. This observation may be relevant to design of media for primary culture of Type II cells, in which disaturated phospholipid synthesis is used as an index of differentiated function (surfactant production). Linoleate supplementation did not alter growth or size (protein content) of cells of either line and caused a slight increase in accumulation of neutral lipid, in the form of cytoplasmic droplets, in A549 cells. Supplementation of cell cultures with equivalent concentrations of the nonessential fatty acids palmitic and oleic acid did not significantly alter the growth, morphologic appearance, or lipid composition of the cells. However, it was demonstrated in cells of one line that palmitic acid supplementation temporarily stimulated synthesis of disaturated choline phosphoglyceride from radiolabeled choline. This work was supported by Grants HL-24817 and HL-21251 from the National Institutes of Health, USPHS, and by a grant from the Alexandrine and Alexander L. Sinsheimer Fund.     

Altered low density lipoprotein receptor regulation is associated with cholesteryl ester accumulation in simian virus 40 transformed rodent fibroblast cell lines

Summary Several SV40 transformed REF52 cell lines were found to accumulate 5 to 10 times more cholesteryl esters compared to their parent line REF52 when cultured in 10% serum. Under this culture condition, the cholesteryl ester to phospholipid ratio was 0.4∶1 and 2.0∶1 to 3.8∶1 for normal and SV40 tranformed cells, respectively. The mechanism underlying cholesteryl ester accumulation in SV40 transformed lines was investigated. We found that 1) the rate of thede novo cholesterol and cholesteryl ester synthesis was roughly equal in normal and transformed derivatives; 2) the accumulation of cholesteryl esters in the transformants would not occur when cultured in lipoprotein-deficient medium and reappeared upon their return to low density lipoprotein-containing medium; 3) the transformants expressed twice as many low density lipoprotein receptors and were less sensitive to LDL-induced receptor down regulation compared to their nontransformed counterparts. The results indicate that SV40 transformed lines exhibited an accelerated lipid uptake from the culture medium due to an altered regulation of low density lipoprotein receptor activity. Supported by NCI grant CA 38016 Editor's Statement This article reports alteration of cholesterol metabolism in rat cells by viral transformation and provides an explanation for the phenomenon at a molecular level. The results may be widely applicable to other instances of viral transformation or virus infection.     

A high level of cell surface phosphatidylinositol-specific phospholipase C activity is characteristic of growth-arrested 3T3 fibroblasts but not of transformed variants.

Confluent monolayers of four contact-inhibited mouse fibroblast lines (Swiss 3T3, Balb/c 3T3, NIH 3T3, and C3H10T1/2) were found to have substantial levels of a cell surface phosphatidylinositol-specific phospholipase C (ecto-PLC). In contrast, confluent cultures of virally, chemically, or spontaneously transformed variants derived from these cell lines expressed undetectable or negligible levels of this enzyme activity. A simple and rapid assay, using lysophosphatidylinositol radio-labeled in the inositol group ([3H]-lysoPI) as the substrate was developed to provide a quantitative measure of the phospholipase C activity present at the external cell surface. For cells testing positive for ecto-PLC activity, rapid uptake of [3H]-lysoPI is accompanied by the simultaneous appearance of [3H]-inositol phosphate in the external medium. Confluent monolayers of the four mouse fibroblast lines exhibiting density-dependent growth inhibition had levels of ecto-PLC activity in the range of 50-800 pmol/min/10(6) cells (i.e., about 20-50 times greater than the activity observed for the transformed variants). The expression of ecto-PLC activity at the cell surface of the Swiss or Balb/c cells was dependent on the state of cell proliferation. Cultures which had become quiescent through attainment of confluence displayed a tenfold increased activity over that of subconfluent, growing cultures of these cells. Similarly, subconfluent Swiss 3T3 cells which had become quiescent following exposure to low serum conditions also showed increased activity. These results indicate that there may exist a correlation between the control of cell proliferation in contact-inhibited mouse fibroblasts and the expression of inositol phospholipid-specific phospholipase C activity at the external cell surface.