Treatment of severe atopic dermatitis by topical immune modulation using dinitrochlorobenzene.

We present the results of a small uncontrolled pilot trial which suggest that contact sensitization to dinitrochlorobenzene and repeated weekly applications significantly improve the clinical status of severe atopic dermatitis in adults. Although no changes were noted in circulating levels of either lymphocyte subset populations or the serum cytokines assayed in this trial, our observations may be due to topical immune modulation by dinitrochlorobenzene. Larger controlled studies of dinitrochlorobenzene treatment in atopic dermatitis are warranted.

Inhibition of neoplastic transformation and bioavailability of dietary flavonoid agents.

Evaluation of unknown biological effects of chemicals including food plant products requires the assessment of bioactivity and bioavailability. Epidemiologic studies show consistently a cancer protective effect of fruit and vegetable consumption, but there is little understanding of which phytochemicals account for this observation. Commonly studied antioxidant micronutrients are less consistently correlated with cancer protection relative to the food groups themselves, suggesting that other phytochemicals or a combination of food products play key roles in preventing cancer. We investigated the effects of the predominant dietary flavonoids and isoflavonoids at inhibiting neoplastic transformation induced by 3-methylcholanthrene in C3H 10T1/2 murine fibroblasts. We found that most phenolic agents tested were equal to or superior to known chemopreventive agents such as carotenoids or vitamins in effectiveness. Hesperetin, hesperidin and catechin were the most potent agents among the flavonoids tested, inhibiting transformation completely when applied at 1.0 microM after exposure to the carcinogen. Structure-activity comparison revealed that among the compounds tested, flavonoids with a vicinal diphenol structure in ring 'B' and a saturated 'C' ring exhibited the strongest effects. Most agents tested showed dose-dependent patterns. Interestingly, the soy isoflavonoids were weakly active except when applied in combination, suggesting a synergistic effect. In addition, HPLC techniques were developed for determining the bioavailability of isoflavonoids in human biological fluids including urine, plasma and breast milk. We observed a relatively fast absorption, distribution and elimination of isoflavonoids including a biphasic pattern probably due to enterohepatic circulation. Total peak isoflavone levels in urine, plasma and in breast milk were found to be 60 microM, 2 microM and 0.2 microM, respectively and were reached 8-12 hours after consumption of soy foods. Levels detected in human body fluids were found to be highly active at inhibiting neoplastic transformation, especially considering synergistic effects observed for combinations of daidzein and genistein, the predominant isoflavonoids occurring in soy foods.

PDGF-stimulated calcium influx changes during in vitro cell transformation.

Platelet Derived Growth Factor (PDGF)-stimulated intracellular calcium signals were obtained from preneoplastic clones derived from C3H 10T1/2 mouse fibroblasts during a four-week transformation assay. By the end of the assay, when transformed colonies of cells were becoming apparent, the PDGF-stimulated calcium signal had reduced significantly. In addition, whole-cell patch-clamp electrophysiology measurements indicated a marked reduction in expression of T-type calcium channels. These observations demonstrate that PDGF-induced calcium metabolism changes as initiated cells progress to the transformed phenotype.

Products of gamma-tocopherol reaction with NO2 and their formation in rat insulinoma (RINm5F) cells.

gamma-Tocopherol, commonly found in seed oils, is the major tocopherol in the U.S. diet, is superior to alpha-tocopherol in preventing neoplastic transformation, and demonstrates unique reactivity toward NO2. This article describes the products of reaction between gamma-tocopherol and low concentrations of gaseous nitrogen dioxide (NO2), as well as their endogenous formation in NO-producing RINm5F cells. gamma-Tocopherol in hexane reacts with NO2 to yield two products identified as 2,7,8-trimethyl-2(4,8,12-trimethyltridecyl)-5,6-chromaquinone++ +, "tocored," and 2,7,8 trimethyl-2(4,8,12-trimethyltridecyl) 5-nitro, 6-chromanol, "tocoyellow." Physical data for these two compounds and reaction characteristics are described. The formation of tocored is consistent with a proposed mechanism of gamma-tocopherol-mediated reduction of NO2 to NO involving initial reaction by NO2 at the C-5 position to form an intermediate nitrite ester tocopheryl radical, which then reacts internally to release NO and form 5,6 epoxy gamma-tocopherol. Tautomerization and further oxidation of the latter intermediate by NO2 yields tocored as the main product observed. The reaction of gamma-tocopherol with NO2 to form NO occurs independently of light, whereas alpha-tocopherol requires light to generate NO from NO2. gamma-Tocopherol and aminoguanidine, an NO synthase inhibitor, were superior to alpha-tocopherol in preventing RINm5F cell toxicity induced by Interleukin-1 beta (IL-1 beta). Both tocored and tocoyellow were observed to form in RINm5F cells loaded with gamma-tocopherol and producing NO constitutively, although a consistent increase in these products as a result of induced NO synthesis was not observed.

Ca2+ influx via T-type channels modulates PDGF-induced replication of mouse fibroblasts.

The role of low-threshold voltage-gated calcium channels (VGCC) in modulating extracellular calcium influx and proliferation was investigated in platelet-derived growth factor (PDGF)-stimulated C3H/10T1/2 mouse fibroblasts. Previous studies demonstrated that cell cycle progression after PDGF stimulation was dependent on extracellular calcium influx producing a sustained increase in the intracellular calcium concentration. In this study, PDGF-induced calcium influx, the sustained intracellular calcium increase, and progression to S phase were inhibited by nordihydroguariaretic acid (NDGA), an inhibitor of calcium influx through VGCC. With the use of the whole cell patch-clamp technique to measure calcium currents, NDGA inhibited inward calcium current through low-threshold VGCC, the only VGCC expressed in C3H/10T1/2 fibroblasts. The inhibitory effects of NDGA on calcium influx and cell proliferation each had a mean inhibitory dose of 2-3 microM. Although NDGA also effectively inhibits cyclooxygenase and lipoxygenase, the addition of prostaglandins or leukotrienes could not reverse this inhibition nor could it be replicated by other antioxidants. These data support the hypothesis that low-threshold VGCC can mediate extracellular calcium influx on the stimulation of cell proliferation by PDGF.

Inhibitors of endogenous nitrogen oxide formation block the promotion of neoplastic transformation in C3H 10T1/2 fibroblasts.

The endogenous production of nitric oxide (NO) and its role in the neoplastic transformation of C3H 10T1/2 mouse fibroblasts were investigated. NO production, as indicated by NO2- in the culture medium, was increased in cells initiated with 3-methylcholanthrene or stimulated with the combination of interferon-gamma (IFN gamma, 10 ng/ml) plus bacterial lipopolysaccharide (LPS, 1 micrograms/ml). NO2- was detectable within 24-48 h of IFN gamma/LPS treatment and accumulated to micromolar concentrations within 4 days. NO production was inhibited in a dose-dependent manner by analogs of L-arginine in which the terminal guanidino nitrogen is blocked, consistent with NO production by the oxidative deamination of L-arginine by nitric oxide synthase (NOS). IFN gamma/LPS-stimulated cells expressed a 4.4 kb mRNA which hybridized to a probe for the mouse macrophage-inducible NOS. Expression of the rat cerebellar constitutive NOS was not detected in these cells. Arginine analogs added to the culture medium during the post-confluence promotional stages of the C3H 10T1/2 transformation assay blocked the formation of transformed foci in a dose-dependent manner comparable to their inhibition of NO production. These data demonstrate that C3H 10T1/2 mouse fibroblasts are a useful model for the study of the effects of endogenous NO production in carcinogenesis and suggest that NO plays a significant role in the promotional phase of neoplastic transformation of these cells.

Competence induction by PDGF requires sustained calcium influx by a mechanism distinct from storage-dependent calcium influx.

The significance and mechanism of extracellular calcium influx in the stimulation by PDGF of cell replication was investigated in density-arrested C3H 10T1/2 mouse fibroblasts. PDGF consistently stimulated a biphasic increase in the [Ca2+]i composed of a rapid transient release of calcium from intracellular storage sites followed by a sustained elevation, significantly greater than prestimulated levels, which was dependent upon the [Ca2+]e and persisted for at least 1 h. The percentage of cells incorporating [3H]-TdR into DNA after stimulation with PDGF+insulin was closely correlated with the magnitude of the sustained [Ca2+]i increase and to the [Ca2+]e. Selective inhibition of the sustained [Ca2+]i increase, by blocking calcium influx with La3+, completely inhibited progression to S phase without affecting the release of calcium from intracellular storage sites. Progression to S phase was inhibited by La3+ or the omission of added extracellular calcium only during PDGF exposure and not during treatment with insulin. PDGF-induced calcium influx was completely inhibited by La3+ whereas storage-dependent calcium influx (SDCI) induced by thapsigargin was unaffected. Pretreatment with TPA, forskolin, dibutyryl-cAMP, dibutyryl-cGMP, nifedipine, and TMB-8 had no effect on PDGF-induced calcium influx. These data suggest that the induction of replicative competence by PDGF is dependent upon the maintenance of a sustained increase in the intracellular calcium concentration due to the influx of extracellular calcium through a calcium influx pathway distinct from SDCI.

Gamma-tocopherol detoxification of nitrogen dioxide: superiority to alpha-tocopherol.

In the vitamin E group, alpha-tocopherol is generally considered to be the most potent antioxidant with the highest vitamin bioactivity, yet gamma-tocopherol is produced in greater amounts by many plants and is the principal tocopherol in the United States diet. This report describes a fundamental difference in the chemical reactivities of alpha-tocopherol and gamma-tocopherol with nitrogen dioxide (NO2), which leads to the formation of a nitrosating agent from alpha-tocopherol, but not from gamma-tocopherol. Nitric oxide (NO) is a major product of the reaction of gamma-tocopherol with NO2, while alpha-tocopherol reacts with NO2 to form an intermediate tocopheroxide analogue. The biological significance of gamma-tocopherol is suggested by limited epidemiological data as well as the observation that it is a more potent inhibitor than alpha-tocopherol of neoplastic transformation during the postinitiation phase in 3-methylcholanthrene-treated C3H/10T1/2 murine fibroblasts. This latter property suggests the superiority of gamma-tocopherol in a mammalian biological assay and a role for endogenous NO production in promotion of neoplastic transformation.

Expression of voltage-gated calcium channels correlates with PDGF-stimulated calcium influx and depends upon cell density in C3H 10T1/2 mouse fibroblasts.

Platelet derived growth factor (PDGF) mobilizes multiple calcium pools in the C3H 10T1/2 mouse fibroblast, including a sustained influx of extracellular calcium. We have used the whole cell patch-clamp technique to directly test for a role of plasma membrane calcium channels in this influx. Whole-cell patch-clamp recordings revealed a voltage-gated calcium channel with gating properties consistent with a 'T-type' designation. This phenotype of the C3H 10T1/2 fibroblasts was dependent upon the cell density in culture. The fraction of cells expressing calcium channels was low (< 10%) in subconfluent culture but rose to approximately 50% as the cells established a confluent monolayer. The magnitude of the PDGF-stimulated sustained calcium influx component measured using Fura-2 increased in parallel with the expression of calcium current. We interpret these results to support the hypothesis that T-type voltage-gated calcium channels contribute to the PDGF-stimulated intracellular calcium signals in these cells.

Okadaic acid inhibits PDGF-induced proliferation and decreases PDGF receptor number in C3H/10T1/2 mouse fibroblasts.

Okadaic acid is both a potent inhibitor of protein serine/threonine phosphatases and a tumor promoter in the mouse skin model. We have previously shown that at non-toxic nanomolar concentrations okadaic acid reversibly inhibits induction (promotion) by PDGF of transformed cells by the 'complete' and 'two-stage' protocols in the C3H/10T1/2 mouse fibroblast transformation assay. In the present study we have demonstrated that treatment of confluent and proliferatively quiescent C3H/10T1/2 mouse fibroblasts with low doses of okadaic acid inhibits the platelet-derived growth factor (PDGF)-induced mitogenic response. This inhibition is accompanied by a loss of PDGF binding sites, a decreased PDGF-induced phosphatidylinositol turnover and a decrease in the PDGF-induced intracellular calcium signal. The decrease in the PDGF-generated intracellular signalling processes represents a mechanism by which okadaic acid inhibits PDGF-induced proliferation and the promotion of in vitro neoplastic transformation by PDGF.

Retinyl acetate inhibits platelet-derived growth factor-induced Ca2+ signals in C3H 10T1/2 fibroblasts.

Mitogenic stimulation of density-arrested C3H 10T1/2 mouse fibroblasts by serum or purified platelet-derived growth factor (PDGF) was potently inhibited by retinyl acetate (RAc; IC50 = 0.1 microgram/ml, 0.3 x 10(-6) M) when administered during the first 2 hours of mitogen exposure. This inhibitory effect of RAc coincided with a period early in the cell growth-division cycle when density-arrested C3H 10T1/2 cells stimulated by PDGF were found to require physiological levels of extracellular Ca2+ for the transition from G0 to G1 of the cell cycle. To determine if the inhibitory effect of RAc was mediated through alterations in the Ca2+ signaling pathway induced by mitogens, we examined Fura-2-loaded fibroblasts for changes in the Ca2+ response elicited by PDGF. Addition of PDGF (5 ng/ml) induced a transient increase in the [Ca2+]i that was not significantly effected by the extracellular Ca2+ concentration. Treatment of cells with RAc caused a concentration- and time-dependent inhibition of this PDGF-stimulated Ca2+ flux (IC50 = 0.45 microgram/ml or 1.5 x 10(-6) M; t1/2 = 15 min), whereas release of intracellularly stored Ca2+ by thrombin was unaffected by RAc (1.2 micrograms/ml, 4 x 10(-6) M). Treatment with RAc did not significantly affect PDGF binding to cell surface receptors or the generation of inositol phosphates. These results suggest that the mechanism by which RAc inhibits PDGF- or serum-induced mitogenesis is through modulation of the Ca2+ signal stimulated by PDGF, and thereby depriving the cell of a rise in intracellular Ca2+ necessary for progression through the cell cycle.

PDGF-induced activation of phospholipase C is not required for induction of DNA synthesis.

Platelet-derived growth factor (PDGF) induction of DNA synthesis is believed to involve activation of phospholipase C (PLC) and subsequent accumulation of inositol 1,4,5-triphosphate [I(1,4,5)P3], increase in intracellular Ca2+, activation of protein kinase C (PKC), and receptor down regulation. Generation of these events is triggered by the tyrosine protein kinase (TPK) activity of the PDGF receptor. The TPK inhibitor genistein blocked PDGF induction of these events, including DNA synthesis, with the exception of receptor down regulation. PDGF-induced phosphotyrosine phosphorylations, including receptor autophosphorylation, were inhibited by genistein. Removal of genistein and PDGF resulted in DNA synthesis without the occurrence of PLC activation. These findings indicate that these early events, with the exception of receptor down regulation, are not necessary for PDGF-induced DNA synthesis.

Okadaic acid: a reversible inhibitor of neoplastic transformation of mouse fibroblasts.

Okadaic acid (OA) is a potent inhibitor of serine/threonine-specific protein phosphatases types 1 and 2A at nanomolar concentrations in cell-free assays and has tumor promoting activity in vivo. We have found that at non-toxic, nanomolar concentrations, OA concentration dependently inhibits the induction of focus-forming transformed cells by the "complete" and "two-stage" protocols in the C3H/10T1/2 mouse fibroblast transformation assay. This inhibitory effect was fully reversible upon removal of OA from the culture medium of carcinogen-treated cells, indicating that OA was not selectively toxic to initiated or transformed cells. Additional treatment with the phorbol ester tumor promoter, TPA, was required to promote the induction of transformed cells after the removal of OA in the two-stage transformation assay. At concentrations that inhibited neoplastic transformation, OA inhibited a type 2A-like phosphohistone protein phosphatase in homogenates of C3H/10T1/2 cells. It is postulated that OA inhibited an early protein phosphatase-sensitive event in the process of in vitro neoplastic transformation by C3H/10T1/2 fibroblasts and had the effect of maintaining carcinogen-treated cells in an initiated state.

Inhibition by retinoids of platelet growth factor-dependent stimulation of DNA synthesis and cell division in density-arrested C3H 10T1/2 fibroblasts.

The inhibition of neoplastic transformation by vitamin A and its natural and synthetic analogues, collectively called retinoids, is accomplished by an as yet unknown mechanism. In a recent report, the morphological transformation of carcinogen-treated C3H 10T1/2 fibroblasts to focus-forming transformed cells was shown to be a postconfluence event induced in density-arrested initiated cells by platelet growth factors in serum and was correlated with the mitogenic response of the preneoplastic cells to these polypeptides. The current study investigates the possibility that the inhibition of neoplastic transformation by retinoids is accomplished by blocking the mitogenic response of initiated cells to these growth factors. The results demonstrate that the stimulation by serum of DNA synthesis and cell division in normal and carcinogen-treated C3H 10T 1/2 fibroblasts after density-dependent growth arrest is inhibited in a dose-dependent manner by retinyl acetate, all-trans retinoic acid, and 4-hydroxyphenyl-retinamide over the same dose range and to the same extent that neoplastic transformation is inhibited by these retinoids. Cellular mitogenic processes sensitive to retinoid inhibition were shown to be induced specifically by platelet growth factors, rather than plasma growth factors, to occur within approximately 2 h of growth factor treatment, and to be common to cell division stimulated in density-arrested normal and initiated cells by highly purified platelet-derived or epidermal growth factor. These data suggest that the inhibition of neoplastic transformation by retinoids is accomplished by blocking the G0 to G1 transition in the mitotic response of initiated cells to platelet growth factors which act as endogenous promoters of transformation.

Induction by growth factors from platelets of the focus-forming transformed phenotype in carcinogen-treated C3H/10T1/2 fibroblasts.

Wounding of mouse skin promotes tumor formation as effectively as 12-O-tetradecanoylphorbol-13-acetate (TPA). Like wounding, TPA stimulates the release of growth factors from platelets and the leakage of plasma from the capillary circulation. Thus, initiated cells in TPA-treated skin are also exposed to the components of whole blood-derived serum. It is possible that serum factors play an important role in the multi-step process of neoplastic transformation. In this study, we evaluate the significance of serum components derived from plasma and platelets in the neoplastic transformation of C3H/10T1/2 mouse fibroblasts exposed to methylcholanthrene. Carcinogen-treated cultures grown to confluence in medium containing 5% whole blood-derived serum (FBS), but maintained for 5 weeks after confluence in plasma-derived serum (PDS), which lacks the platelet components found in whole blood-derived serum, failed to produce transformed foci. The addition of an aqueous extract of platelets to PDS induced the formation of transformed foci with an efficiency comparable to FBS and proportional to the amount and mitogenic activity of the platelet extract. The growth of newly transformed cells was not inhibited by the absence of platelet factors in the culture medium. The loss of density-dependent growth control required at least 3-4 weeks of post-confluence exposure to serum factors derived from platelets. The data suggest that platelet factors induce the conversion of a carcinogen-initiated cell to a focus-forming transformed cell. We demonstrate that platelet-derived growth factor (PDGF) is essential for the induction of the focus-forming phenotype in initiated cells and that PDGF acts co-operatively with the platelet-derived type-beta transforming growth factor and EGF-like growth factor to induce this transformed phenotype. These growth factors may be acting as endogenous promoters of neoplastic transformation of chemically initiated C3H/10T1/2 mouse fibroblasts.

Growth of kidney epithelial cells in culture: evidence for autocrine control.

The factors that stimulate kidney growth in K+-deficient animals are unknown. Cultures of renal epithelial cells (BSC-1 line) were used to study this phenomenon because their growth is accelerated in medium containing a reduced K+ concentration. We tested the hypothesis that growth induced by low-K+ medium is mediated by factors produced by the cells; i.e., is subject to autocrine control. Low-K+ (3.2 mM) or control (5.4 mM) medium was conditioned by placing it on confluent cultures of BSC-1 cells for 1 h and was then collected. The K+ concentration of the low-K+ conditioned medium was then adjusted to the control value by addition of KCl. This conditioned medium stimulated growth of fresh cultures of cells to the same extent as did unconditioned low-K+ medium. The appearance of growth-promoting activity was maximal at a K+ concentration of 3.2 mM during conditioning of the medium. Low-K+ conditioned medium, corrected to a K+ concentration of 5.4 mM, required 6 h to commit cells to enhanced proliferation. Growth-stimulating activity in low-K+ conditioned medium was antagonized by a purified growth inhibitor produced by the cells. These observations are consistent with the hypothesis that autocrine products with opposite effects on growth can regulate proliferation of renal epithelial cells.

Modulation by retinyl acetate of microfilament bundle formation in C3H/10T1/2 cells.

Retinyl acetate has been previously shown to inhibit carcinogen-induced neoplastic transformation in 10T1/2 cells and to accentuate many aspects of the nontransformed phenotype. Scanning electron microscopy of logarithmic phase 10T1/2 cells treated for 3 days with 0.3 micrograms/ml retinyl acetate revealed that this treatment caused extensive flattening of cells to the plastic substrate. In contrast the tumor promoter tetradecanoyl phorbol acetate, which antagonizes the antineoplastic activity of retinyl acetate, caused cell rounding and completely inhibited the action of retinyl acetate on cell morphology. During this same time course, the formation of microfilament bundles was also found to be modulated by retinyl acetate. Transmission electron micrographs of unsectioned peripheral regions of flattened cells showed that while the unit density of microfilament bundles was not influenced, the thickness of bundles, particularly those with a diameter of 100 nm or more, was increased by retinyl acetate. Tetradecanoyl phorbol acetate had little effect on microfilament bundle diameters but did partially antagonize the action of retinyl acetate. To determine if this increase was associated with an increase in total actin/cell, total cell proteins, and proteins not extractable by glycerol-triton extraction, were subjected to sodium dodecylsulfate/ polyacrylamide gel electro-phoresis. It was found that while total cellular actin was not increased by retinyl acetate, the proportion of nonextractable actin (which includes microfilament bundles) increased from 65% to 88% of total actin. This increase was not inhibited by inhibitors of protein or RNA synthesis. These studies again demonstrate that retinyl acetate accentuates the nontransformed phenotype of 10T1/2 cells; it is hypothesized that these actions are related to the antineoplastic activity of retinoids.

Quantitative neoplastic transformation of C3H/10T1/2 fibroblasts: dependence upon the size of the initiated cell colony at confluence.

The efficiency of transformation of C3H/10T1/2 fibroblasts by chemical or physical carcinogens varies inversely with the seeding density of the cells. Colony size at confluence, accumulated cell generations, and epigenetic events independent of seeding density have been proposed to explain this variability. By controlling the total number and colony size (i.e., cells/colony) of initiated cells at confluence and their accumulated cell generations, we have determined that (a) the colony size of the initiated cells at confluence is directly related to the expression of the transformed phenotype, (b) the probability that an initiated cell will form a colony of transforming cells does not vary with the accumulation of large numbers of cell generations, and (c) the total number of initiated cells in a confluent culture does not determine the number of transformed foci formed. We have developed a mathematical representation which, when applied to our results and the published results from other laboratories, clearly describes the relationship between the size of the initiated cell colony at confluence and the expression of the transformed phenotype. These results have significant implications in the quantification of the transformation of C3H/10T1/2 cells by chemical and physical carcinogens.

Retinoid effects on cell-cell interactions and growth characteristics of normal and carcinogen-treated C3H/1OT1/2 cells.

The effects of retinyl acetate and all-trans-retinoic acid on the growth rate, saturation density, and cytoplasmic underlapping of normal and carcinogen-initiated C3H/1OT1/2 cells were examined. Retinyl acetate (a) decreased the saturation density by as much as 45%, (b) had no effect on the growth rate, (c) reduced cytoplasmic underlapping of adjacent cells by 55 to 85%, and (d) inhibited neoplastic transformation by methylcholanthrene. The effects were dose dependent and not significantly affected by the serum concentrations over the range of 2.5 to 10%. In contrast, all-trans-retinoic acid (a) decreased the saturation density as effectively as retinyl acetate, but only in medium containing 10% serum; (b) significantly reduced the growth rate of cells at low density, especially at low serum concentrations; (c) had no effect upon cytoplasmic underlapping; and (d) enhanced transformation at nontoxic concentrations in medium containing 5% serum but had no effect in 10% serum. We conclude that the effects of retinoids on the growth rate and saturation density of cells in culture may not be relevant to their inhibition of neoplastic transformation. Rather, we interpret the results of our experiments on cytoplasmic underlapping as an indication that retinoids inhibit transformation by stabilizing and/or enhancing cell surface receptors involved in cell-cell contact-dependent formation of a stable monolayer.

Isolation of methylcholanthrene-"initiated" C3H/10T1/2 cells by inhibiting neoplastic progression with retinyl acetate.

A clone of 3-methylcholanthrene-treated 10T1/2 cells has been isolated which possesses basic characteristics expected of "initiated" cells. In the presence of retinyl acetate, this clone exhibits contact inhibited growth control and is morphologically indistinguishable from the parental 10T1/2 cell line. Removal of retinyl acetate in vitro results in neoplastic transformation after a latent period of 3 weeks. The classical 10T1/2 transformation system was reconstructed by coculturing normal and "initiated" 10T1/2 cells formed either Type II or Type III foci after a latent period of 3-4 weeks, and an additional 22% formed Type I foci. Treatment of "initiated" 10T/2 cells with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate accelerated the formation of transformed foci in the coculture system by reducing the length of the latent period to less than 3 weeks. Injection of 10(6) "initiated" cells/mouse s.c. into nude mice resulted in the appearance of progressively growing fibrosarcomas after a latent period of 5-7 weeks. Dietary supplementation with 4-hydroxyphenyl-retinamide prevented tumor formation; after drug withdrawal, tumors developed in all surviving mice after 6 weeks. We believe this cell line possesses all characteristics expected of "initiated" cells. With this new cell line, designated INIT/10T1/2, we can now study the early biochemical changes in growth control mechanisms resulting in neoplastic transformation and the mechanism(s) of chemoprevention of cancer by vitamin A.