Non-proliferative State Research Articles

DNA microarray analysis of gene expression in endothelial cells in response to 24-h shear stress.

The recently developed DNA microarray technology provides a powerful and efficient tool to rapidly compare the differential expression of a large number of genes. Using the DNA microarray approach, we investigated gene expression profiles in cultured human aortic endothelial cells (HAECs) in response to 24 h of laminar shear stress at 12 dyn/cm(2). This relatively long-term shearing of cultured HAECs led to the modulation of the expression of a number of genes. Several genes related to inflammation and EC proliferation were downregulated, suggesting that 24-h shearing may keep ECs in a relatively noninflammatory and nonproliferative state compared with static cells. Some genes were significantly upregulated by the 24-h shear stress; these includes genes involved in EC survival and angiogenesis (Tie2 and Flk-1) and vascular remodeling (matrix metalloproteinase 1). These results provide information on the profile of gene expression in shear-adapted ECs, which is the case for the native ECs in the straight part of the aorta in vivo.

Induction of Atypical Ductal Hyperplasia in Mouse Mammary Gland Organ Culture

Induction of Atypical Ductal Hyperplasia in Mouse Mammary Gland Organ Culture

MRG 15, A Chromodomain Protein Related to Mortality Factor 4 (MORF 4) is Involved in Multiple Complexes.

INTRODUCTION. Replicative senescence, the final non-proliferative state reached by normal cells when cultured in vitro, is considered a model of aging process at the cellular level. Senescence is dominant over indefinite division (1) and analysis of hybrids obtained by fusion of immortal human cell lines has defined four complementation groups (A-D) for indefinite division (2). Genes responsible for senescence are located on human chromosomes 1, 4 and 7 for complementation groups C, B and D, respectively. The mortality factor 4 (MORF 4) gene, cloned in our laboratory, is on chromosome 4 and is capable of inducing senescence in cell lines from group B with no effect on cell lines from the other groups (3). Two MORF 4 related genes, MRG X and MRG 15, have been cloned. These genes are of interest as they are induced by serum stimulation. The three proteins have common features, nuclear localization signal, helix-loop-helix and leucine zipper motifs. MRG 15 is of particular interest, as it has an Nterminal extension harboring a chromodomain. Using the yeast two-hybrid system a novel protein, PAM (protein associated with MRG), has been identified as an interacting partner of MRG 15 (Leung and Pereira Smith, unpublished results). We have determined that there are at least two different nucleoprotein complexes that involve MRG 15 and identified some of the interacting partners. METHODS. Stable clones expressing HA-tagged versions of MRG 15 and PAM following transfection of EJ cells were isolated for these studies. Fractionation of nucleoprotein complexes was done by sedimentation on 5 20% sucrose gradients. Fractions were analyzed by Western blot using an anti-HA antibody (Santa Cruz Biotechnology, Inc.) or an antibody raised against a peptide from the N-terminal extension of MRG 15. The same nuclear extracts were analyzed by IP-Western to assess the interactions. GFP-tagged versions of native MRG 15 and deletion mutants lacking the chromodomain or the leucine zipper region were transfected into the stable-expressing PAM-HA clone using Lipofectamine plus (GIBCO BRL products) according to manufacturer instructions. Nucleoprotein complexes containing the native and mutant MRG 15 proteins were analyzed by sucrose gradients, and colocalization determined by confocal microscopy. RESULTS. Analysis of fractions from sucrose gradients of nuclear extracts from cells stablyexpressing MRG 15-HA have detected two protein complexes of 3.5 and 14 S. PAM is associated with the 3.5 S complex. The studies with deletion mutants indicate that the leucine

Hypoxic cytotoxic agents: a new approach to cancer chemotherapy.

Many of the cells in solid tumors are hypoxic. This makes them resistant to killing by both radiotherapy and most commonly used anticancer drugs. The resistance towards anticancer drugs is because hypoxia induces a non-proliferative state as well as the fact that the hypoxic cells are furthest from the blood vessels in the tumor (and hence the source of drug). However, a new class of drugs - hypoxic cytotoxins can, by killing these hypoxic cells, not only overcome this resistance, but also exploit hypoxia thereby converting it into a therapeutic advantage. The first drug to be tested in the clinic purely for its activity towards hypoxic cells, tirapazamine, is described. Copyright 2000 Harcourt Publishers Ltd.

Angiogenesis, metastasis, and endogenous inhibition.

Angiogenesis and the development of metastases are intrinsically connected. Experimental data suggest that establishment and growth of metastases are influenced by soluble factors secreted from the originating solid tumor. Among these factors are so-called endogenous inhibitors of angiogenesis which keep metastasis in a non-proliferating quiescent state. For a number of tumors it has been shown that this dormant state is mediated through inhibition of angiogenesis. This dormant state is characterized by normal proliferation, increased apoptosis, and insufficient neo-vascularization. Removal of inhibiting anti-angiogenic factors led to growth of dormant metastases. A number of endogenous inhibitors have been identified and have shown success in experimental therapeutic trials. This might be of special interest for the treatment of cerebral metastases which are the most common type of malignant brain tumors. Similar to the spread of metastases, it is known that single glioma cells can be found in distant parts of the brain. While local recurrence is a common phenomenon in glioma, formation of clinical apparent distant metastasis occurs rarely. Several lines of evidence suggest that growth inhibition of remote glioma cells may be mediated by an endogenous inhibitory mechanism.

Differentiation between Senescence (M1) and Crisis (M2) in Human Fibroblast Cultures

Normal human fibroblasts undergo only a limited number of divisions in culture and eventually enter a nonreplicative state designated senescence or mortality stage 1 (M1). Expression of certain viral oncogenes, such as the SV40 large T antigen (SV40 T-Ag), can elicit a significant extension of replicative life span, but these cultures eventually also cease dividing. This proliferative decline has been designated crisis or mortality stage 2 (M2). BrdU incorporation assays are commonly used to distinguish between senescence (<5% labeling index) and crisis (>30% labeling index). It has not been possible, however, to ascertain whether the high labeling index, indicative of ongoing DNA replication, was caused by the presence of T-Ag. We used gene targeting to knock out both copies of the p21CIP1/WAF1 gene in presenescent human fibroblasts. p21 −/− cells displayed an extended life span but eventually entered a nonproliferative state. In their terminally nonproliferative state both p21 +/+ and p21 −/− cultures were positive for the senescence-associated β-galactosidase (SA-β-gal) activity; in contrast, the labeling index of p21 +/+ cells was low (<5%) whereas the labeling index of p21 −/− cells was high (>30%). The observation that p21 −/− and SV40 T-Ag-expressing cells behave identically with respect to life span extension as well as the high labeling index in the terminally nonproliferative state indicates that crisis is not a phenomenon induced solely by viral oncogenes, but a physiological state resulting from the bypass of normal senescence mechanisms. The widely used biomarker for senescence, SA-β-gal, cannot distinguish between senescence and crisis. We propose that all SA-β-gal-positive cultures should be further examined for their BrdU labeling index.

Proliferating Cell Nuclear Antigen in Normal and Regenerating Rat Livers

Immunohistochemical analysis using proliferating cell nuclear antigen (PCNA) antibody shows a negligible number of cells stained in normal liver, but much higher numbers in regenerating liver 24 and 48 h after surgery. We also verified different results by biochemical analysis. Two forms of PCNA, L type (eluted at low concentrations of KCl from a phosphocellulose column) and H type (eluted at high KCl concentrations), were observed in the nucleoplasm of regenerating livers 24 and 48 h after surgery. Treatment of the H type fraction with nuclease caused the H type to disappear and the amount of L type to increase. PCNAs in the cytoplasm are P type (eluted in the pass through fraction) and L type. Surprisingly, the total amounts of P type and L type in cytoplasmic extracts are comparable to those of L type and H type in the nucleoplasm. These results suggest that newly synthesized PCNA is immediately converted into the P and L complex forms. The P type and some of the L type that lacks a nuclear localization signal remain in the cytoplasm; the rest of the L type with a nuclear localization signal is transferred into the nuclei. Then, some of the L type in the nucleoplasm forms the H type, which binds to DNA. These three types of PCNA are also found in significant amounts in the nucleoplasm and cytoplasm of normal rat liver despite its nonproliferating state.

P16(INK4a) and the control of cellular proliferative life span.

Normal somatic cells have a limited proliferative capacity in vitro: after a finite number of cell divisions they eventually enter a non-proliferative state referred to as senescence. Senescence is thought to be a major tumor suppressor mechanism, and many cancers contain cells that have escaped from senescence and become immortalized. The role of telomerase activation in immortalization is currently attracting considerable attention, but immortalization is often associated with other changes including loss of normal function of the tumor suppressor locus, INK4a/ARF. Two proteins, p16(INK4a) and p14(ARF), are encoded by this locus. Here we focus on p16(INK4a) and review accumulating evidence that loss of p16(INK4a) function may be involved in escape from the normal limits on cellular proliferative life span.

P27Kip1 links cell proliferation to morphogenesis in the developing organ of Corti

Strict control of cellular proliferation is required to shape the complex structures of the developing embryo. The organ of Corti, the auditory neuroepithelium of the inner ear in mammals, consists of two types of terminally differentiated mechanosensory hair cells and at least four types of supporting cells arrayed precisely along the length of the spiral cochlea. In mice, the progenitors of greater than 80% of both hair cells and supporting cells undergo their terminal division between embryonic day 13 (E13) and E14. As in humans, these cells persist in a non-proliferative state throughout the adult life of the animal. Here we report that the correct timing of cell cycle withdrawal in the developing organ of Corti requires p27(Kip1), a cyclin-dependent kinase inhibitor that functions as an inhibitor of cell cycle progression. p27(Kip1) expression is induced in the primordial organ of Corti between E12 and E14, correlating with the cessation of cell division of the progenitors of the hair cells and supporting cells. In wild-type animals, p27(Kip1) expression is downregulated during subsequent hair cell differentiation, but it persists at high levels in differentiated supporting cells of the mature organ of Corti. In mice with a targeted deletion of the p27(Kip1) gene, proliferation of the sensory cell progenitors continues after E14, leading to the appearance of supernumerary hair cells and supporting cells. In the absence of p27(Kip1), mitotically active cells are still observed in the organ of Corti of postnatal day 6 animals, suggesting that the persistence of p27(Kip1) expression in mature supporting cells may contribute to the maintenance of quiescence in this tissue and, possibly, to its inability to regenerate. Homozygous mutant mice are severely hearing impaired. Thus, p27(Kip1) provides a link between developmental control of cell proliferation and the morphological development of the inner ear.

Ras Proteins Induce Senescence by Altering the Intracellular Levels of Reactive Oxygen Species

Human diploid fibroblasts eventually lose the capacity to replicate in culture and enter a viable but nonproliferative state of senescence. Recently, it has been demonstrated that retroviral-mediated gene transfer into primary fibroblasts of an activated ras gene (V12ras) rapidly accelerates development of the senescent phenotype. Using this in vitro system, we have sought to define the mediators of Ras-induced senescence. We demonstrate that expression of V12Ras results in an increase in intracellular and in particular, mitochondrial reactive oxygen species. The ability of V12Ras to induce growth arrest and senescence is shown to be partially inhibited by coexpression of an activated rac1 gene. A more dramatic rescue of V12Ras-expressing cells is demonstrated when the cells are placed in a low oxygen environment, a condition in which reactive oxygen species production is inhibited. In addition, in a 1% oxygen environment, Ras is unable to trigger an increase in the level of the cyclin-dependent kinase inhibitor p21 or to activate the senescent program. Under normoxic (20% O2) conditions, the V12Ras senescent phenotype is demonstrated to be unaffected by scavengers of superoxide but rescued by scavengers of hydrogen peroxide. These results suggest that in normal diploid cells, Ras proteins regulate oxidant production and that a rise in intracellular H2O2 represents a critical signal mediating replicative senescence.

RNA Antisense Abrogation of MAT1 Induces G1 Phase Arrest and Triggers Apoptosis in Aortic Smooth Muscle Cells

The human MAT1 gene (ménage à trois 1) is an assembly factor and a targeting subunit of cyclin-dependent kinase (CDK)-activating kinase. The novel mechanisms by which MAT1 forms an active CDK-activating kinase and determines substrate specificity of CDK7-cyclin H are involved in the cell cycle, DNA repair, and transcription. Hyperplasia of vascular smooth muscle cells (SMC) is a fundamental pathologic feature of luminal narrowing in vascular occlusive diseases, and nothing is yet known regarding the cell cycle phase specificity of the MAT1 gene in its involvement in SMC proliferation. To investigate such novel regulatory pathways, MAT1 expression was abrogated by retrovirus-mediated gene transfer of antisense MAT1 RNA in cultured rat aortic SMCs. We show that abrogation of MAT1 expression retards SMC proliferation and inhibits cell activation from a nonproliferative state. Furthermore, we have demonstrated that these effects are due to G1 phase arrest and apoptotic cell death. Our studies indicate a link between cell cycle control and apoptosis and reveal a potential mechanism for coupling the regulation of MAT1 with G1 exit and prevention of apoptosis.

Cyclic Nucleotide Phosphodiesterases and Human Arterial Smooth Muscle Cell Proliferation

IntroductionSmooth muscle cells (SMCs) in the arterial wall are normally found in a contractile, nonproliferative state. These SMCs are continuously exposed to agents that elevate cyclic AMP (cAMP) and cyclic GMP (cGMP), such as prostacyclin and nitric oxide (NO) released from the endothelium. Both cAMP and cGMP potently inhibit SMC proliferation by antagonizing major signaling pathways induced by growth factors, such as platelet-derived growth factor. Different forms of injury to the endothelium result in proliferation of the SMCs and can develop into atherosclerosis, restenosis, or arterial thickening associated with neonatal pulmonary hypertension. In human adult aorta, cAMP and cGMP levels are tightly regulated by synthesis and degradation. The latter is mediated by at least two different cAMP phosphodiesterases (PDEs), PDE3 and PDE4, and three different PDEs that degrade mainly cGMP, PDE1A, PDE1B, and PDE5. Strikingly, expression of a new PDE that degrades both cAMP and cGMP, PDE1C, is markedly induced in proliferating SMCs from the same aorta in culture, whereas its expression is nondetectable in contractile aorta. We propose that, for a human arterial SMC to be able to efficiently proliferate, and perhaps even to undergo a phenotypic change from a quiescent to a proliferative phenotype, it has to efficiently degrade inhibitory cAMP and cGMP. In human SMCs, PDE1C is induced to degrade these cyclic nucleotides. Understanding the role of PDE1C in SMC proliferation may provide the basic information necessary for development of highly specific PDE1C inhibitors that target proliferating SMCs in cardiovascular disease.

Fibroblast growth factor (FGF) soluble receptor 1 acts as a natural inhibitor of FGF2 neurotrophic activity during retinal degeneration.

Fibroblast growth factors (FGF) 1 and 2 and their tyrosine kinase receptor (FGFR) are present throughout the adult retina. FGFs are potential mitogens, but adult retinal cells are maintained in a nonproliferative state unless the retina is damaged. Our work aims to find a modulator of FGF signaling in normal and pathological retina. We identified and sequenced a truncated FGFR1 form from rat retina generated by the use of selective polyadenylation sites. This 70-kDa form of soluble extracellular FGFR1 (SR1) was distributed mainly localized in the inner nuclear layer of the retina, whereas the full-length FGFR1 form was detected in the retinal Muller glial cells. FGF2 and FGFR1 mRNA levels greatly increased in light-induced retinal degeneration. FGFR1 was detected in the radial fibers of activated retinal Muller glial cells. In contrast, SR1 mRNA synthesis followed a biphasic pattern of down- and up-regulation, and anti-SR1 staining was intense in retinal pigmented epithelial cells. The synthesis of SR1 and FGFR1 specifically and independently regulated in normal and degenerating retina suggests that changes in the proportion of various FGFR forms may control the bioavailability of FGFs and thus their potential as neurotrophic factors. This was demonstrated in vivo during retinal degeneration when recombinant SR1 inhibited the neurotrophic activity of exogenous FGF2 and increased damaging effects of light by inhibiting endogenous FGF. This study highlights the significance of the generation of SR1 in normal and pathological conditions.

Increased expression of cyclin D2 during multiple states of growth arrest in primary and established cells.

Cyclin D2 is a member of the family of D-type cyclins that is implicated in cell cycle regulation, differentiation, and oncogenic transformation. To better understand the role of this cyclin in the control of cell proliferation, cyclin D2 expression was monitored under various growth conditions in primary human and established murine fibroblasts. In different states of cellular growth arrest initiated by contact inhibition, serum starvation, or cellular senescence, marked increases (5- to 20-fold) were seen in the expression levels of cyclin D2 mRNA and protein. Indirect immunofluorescence studies showed that cyclin D2 protein localized to the nucleus in G0, suggesting a nuclear function for cyclin D2 in quiescent cells. Cyclin D2 was also found to be associated with the cyclin-dependent kinases CDK2 and CDK4 but not CDK6 during growth arrest. Cyclin D2-CDK2 complexes increased in amounts but were inactive as histone H1 kinases in quiescent cells. Transient transfection and needle microinjection of cyclin D2 expression constructs demonstrated that overexpression of cyclin D2 protein efficiently inhibited cell cycle progression and DNA synthesis. These data suggest that in addition to a role in promoting cell cycle progression through phosphorylation of retinoblastoma family proteins in some cell systems, cyclin D2 may contribute to the induction and/or maintenance of a nonproliferative state, possibly through sequestration of the CDK2 catalytic subunit.

Persistence of peptide-induced CD4+ T cell anergy in vitro.

Clonal T cell unresponsiveness, or anergy, has been proposed as a mechanism of peripheral tolerance in vivo, and as a potential means of curbing unwanted T cell responses. In this study, anergy was induced in a T helper cell (Th) clone reactive to hemoglobin (Hb) peptide 64-76 by coculture of the T cells with live antigen-presenting cells (APCs) and 74L, a peptide analog of Hb(64-76) that contains a single amino acid substitution of leucine for glycine at position 74, or with a low concentration of the agonist ligand. The anergic state was characterized by blunted proliferation and interleukin (IL) 2 production upon restimulation with Hb(64-76), and was not the result of impaired TCR/CD3 downmodulation. The addition of exogenous IL-12 transiently restored proliferation of the anergic lines, but removal of IL-12 from culture returned the T cells to their nonproliferative state. Interestingly, persistence of the anergic phenotype was observed despite biweekly restimulation with antigen, APCs, and IL-2. Thus, T cell unresponsiveness induced by a peptide produced a stable, persistent anergic state in a Th0 clone that was not reversible by stimulation with IL-2 or -12.

Protection provided by exogenous DNA ligase in G0 human lymphocytes treated with restriction enzyme MspI or bleomycin as shown by the comet assay

DNA double-strand breaks (DSB) may arise either spontaneously during cellular processes or as a result of exposure to DNA-damaging agents such as ionizing radiation, or radiomimetic agents such as restriction endonucleases or bleomycin. It is widely accepted that nonrepaired or misrepaired DSB are the main lesions leading to the production of chromosomal aberrations, mutagenesis, oncogenic transformation, and cell killing. Studies focusing on this relationship, as well as the possible modulation of DNA repair mechanisms, are currently of major interest. A wide variety of test systems are available to study DNA damage. In the last few years, single-cell gel electrophoresis, commonly known as "comet assay," has been considered a rapid, sensitive, and visual method for quantifying DNA strand breaks and alkali-labile damage in individual cells. In this study, making use of the comet assay, we tried to find out if under conditions that maintain chromatin structure the DNA ligase from T4 phage is able to facilitate the rejoining of strand breaks with different end structures, induced by the restriction endonuclease MspI or bleomycin in living human lymphocytes in a nonproliferating state. T4 DNA ligase, as well as the restriction endonuclease or bleomycin, were introduced together by electroporation into human lymphocytes. Our results support the idea that it is possible to modulate the DSB-rejoining of different DNA strand-breaking agents by exogenous T4 DNA ligase.

Big brothers are watching: the retinoblastoma family and growth control.

Multicellular organisms consist of numerous types of cells and each of these cells has characteristic properties in its pattern of gene expression, its degree of multiplication rate, its state of differentiation, and its life span. The cellular programs are guided by both inner- and extracellular signals converging onto various molecular pathways. Dependent on this information, the cell decides whether to respond with proliferation, differentiation, quiescence, or apoptosis in gene-directed processes to maintain homeostasis. Loss of genetic integrity affecting either growth-promoting (proto oncogenes) or growth-inhibiting (tumor suppressor genes) sources could be the initial step in the multistep development of neoplasia. While heterozygous mutations of proto oncogenes can be sufficient for cellular transformation, only homozygous mutations of tumor suppressor genes, such as the retinoblastoma protein pRB, have been reported to cause cancer in mammalian cells to date. The tumor suppressor p53 is an exception, as dominant negative mutants have been shown to elicit cellular transformation (Bishop 1991). The majority of cells in a developed mammalian organism are in a non-proliferative, either differentiated or quiescent state, and the commitment to become a particular specialized cell necessitates its withdrawal from the cell cycle. The retinoblastoma protein family members pRB, p 107, and RB2/p 130 have in the past been shown to play a major role for cells to keep these commitments and prevent cells from improperly reentering or improperly staying in the cell-division cycle. In fact, inactivation of the RB family is an integral event for a large number of diverse growth promoting pathways.

Immunosenescence: Analysis and Genetic Modulation of Replicative Senescence in T Cells

Immunosenescence, which constitutes one of the most dramatic physiologic changes associated with aging, may account for the increased susceptibility to infections and the high incidence of cancer in the elderly. A novel facet of T-cell biology has been recently identified that may exert a considerable impact on immune control over infections and cancer during aging. Cell culture studies have shown that after repeated rounds of antigen-driven proliferation, T lymphocytes eventually reach replicative senescence, an irreversible nonproliferative state associated with the loss of expression of a critical T-cell signaling molecule. Identification of this unique, cell-specific marker of senescence has facilitated the documentation and analysis of replicative senescence within the immune system in vivo during aging. This article summarizes the features of T-cell replicative senescence and highlights several genetic strategies that may reverse the process. The ability to manipulate T-cell replicative senescence may ultimately provide a fresh therapeutic approach to extend the years of immunologie "coverage" in the elderly.

Developmental Regulation of Mac25/Insulin-like Growth Factor-Binding Protein-7 Expression in Skeletal Myogenesis

Mac25 is a newly discovered member of the insulin-like growth factor-binding protein (IGFBP) family, recently assigned the name IGFBP-7. Mac25/IGFBP-7 is hypothesized to have growth-suppressing activity, since mac25/IGFBP-7 mRNA is down-regulated in several tumor cell lines and is highly expressed in senescent mammary epithelial cells. In this study, mac25/IGFBP-7 mRNA expression was characterized in the C2 skeletal myogenic cell line, which undergoes a transition from actively dividing, undifferentiated myoblasts to nondividing, differentiated myotubes. Mac25/IGFBP-7 mRNA levels were 2.5-fold higher in dividing C2 myoblasts than in nondividing myotubes. The inverse correlation between mac25/IGFBP-7 expression and myogenic differentiation was further examined by treating myogenic cultures with transforming growth factor-β (TGF-β) or insulin-like growth factor-I (IGF-I). TGF-β inhibited myogenic differentiation by 98% and stimulated mac25/IGFBP-7 mRNA expression 2-fold. IGF-I stimulated differentiation by 50% and inhibited mac25/IGFBP-7 expression 2- to 3-fold. These findings indicate that, in contrast to other cell systems examined so far, expression of this new member of the IGFBP family is not always correlated with a nonproliferative state.

Recovery of Ca2+ Pools and Growth in Ca2+Pool-depleted Cells Is Mediated by Specific Epoxyeicosatrienoic Acids Derived from Arachidonic Acid

Depletion of Ca2+ pools using the irreversible Ca2+ pump blocker, thapsigargin, induces DDT1MF-2 smooth muscle cells to enter a stable nonproliferative state. Reversal of this state can be mediated by high (20%) serum treatment, which induces new Ca2+ pump protein, return of Ca2+ pools, and reentry of cells into the cell cycle; the effect of serum can be mimicked by the essential fatty acids (EFA), arachidonic, linoleic, and alpha-linolenic acids (Graber, M.N., Alfonso, A., and Gill, D.L., (1996) J. Biol. Chem. 271, 883-888). The possible requirement for EFA metabolism in inducing recovery of Ca2+ pool-depleted growth-arrested cells was investigated. Neither cyclooxygenase or lipoxygenase inhibitors had any effect on arachidonic acid-induced growth recovery of thapsigargin-treated cells. In contrast, the cytochrome P-450 epoxygenase inhibitors, SKF525A and metyrapone, substantially reduced arachidonic acid-induced recovery of growth while having minimal effects on control cell growth. Both epoxygenase inhibitors completely prevented the arachidonic acid-induced recovery of bradykinin-releasable Ca2+-pumping pools, whereas cyclooxygenase and lipoxygenase inhibitors had no effect. The effectiveness of the four cytochrome P-450 metabolites of arachidonic acid on recovery of Ca2+ pools were compared; 8,9- and 11,12-epoxyeicosatrienoic acid (EET) at 1.5 microM were completely effective in recovering agonist-sensitive Ca2+ pools, whereas the 5,6- and 14,15-EETs were without effect. SKF525A did not block the action of 8,9- or 11, 12-EET indicating further P-450 metabolism was not required. Hydration of the active EET molecules prevented Ca2+ pool recovery since the dihydroxy-derivatives of both 8,9- and 11,12-EET were ineffective. The specificity of effectiveness among EET molecules for subsequent resumption of growth of thapsigargin-treated cells was the same as for Ca2+ pool recovery. Significantly, the P-450 inhibitors, SKF525A and metyrapone, both prevented the action of 20% serum in inducing recovery of thapsigargin-treated cells, whereas cyclooxygenase and lipoxygenase inhibitors were ineffective, indicating that EFAs are the active component within serum that is responsible for recovery of Ca2+ pool-depleted cells. The specific action of EETs in mediating recovery of Ca2+ pools and growth of thapsigargin-treated cells represents not only a novel action of epoxygenase products from EFAs, but also a potentially significant new signaling pathway that may effect translational control and regulate transition from a stationary to proliferative growth state.