Showing papers on "Cell growth published in 1996"

Cancer Cell Cycles

Abstract: Uncontrolled cell proliferation is the hallmark of cancer, and tumor cells have typically acquired damage to genes that directly regulate their cell cycles. Genetic alterations affecting p16(INK4a) and cyclin D1, proteins that govern phosphorylation of the retinoblastoma protein (RB) and control exit from the G1 phase of the cell cycle, are so frequent in human cancers that inactivation of this pathway may well be necessary for tumor development. Like the tumor suppressor protein p53, components of this "RB pathway," although not essential for the cell cycle per se, may participate in checkpoint functions that regulate homeostatic tissue renewal throughout life.

Inhibition of Adipogenesis Through MAP Kinase-Mediated Phosphorylation of PPARγ

Abstract: Adipocyte differentiation is an important component of obesity and other metabolic diseases. This process is strongly inhibited by many mitogens and oncogenes. Several growth factors that inhibit fat cell differentiation caused mitogen-activated protein (MAP) kinase-mediated phosphorylation of the dominant adipogenic transcription factor peroxisome proliferator-activated receptor γ (PPARγ) and reduction of its transcriptional activity. Expression of PPARγ with a nonphosphorylatable mutation at this site (serine-112) yielded cells with increased sensitivity to ligand-induced adipogenesis and resistance to inhibition of differentiation by mitogens. These results indicate that covalent modification of PPARγ by serum and growth factors is a major regulator of the balance between cell growth and differentiation in the adipose cell lineage.

Inhibition of acute lymphoblastic leukaemia by a Jak-2 inhibitor

Abstract: ACUTE lymphoblastic leukaemia (ALL) is the most common cancer of childhood. Despite the progress achieved in its treatment, 20% of cases relapse and no longer respond to chemotherapy. The most common phenotype of ALL cells share surface antigens with very early precursors of B cells and are therefore believed to originate from this lineage1,3Characterization of the growth requirement of ALL cells indicated that they were dependent on various cytokines, suggesting paracrine and/or autocrine growth regulation4–6. Because many cytokines induce tyrosine phosphorylation in lymphoid progenitor cells, and constitutive tyrosine phosphorylation is commonly observed in B-lineage leukaemias7,8, attempts have been made to develop protein tyrosine kinase (PTK) blockers of leukaemia cell growth9,10. Here we show that leukaemic cells from patients in relapse have con-stitutively activated Jak-2 PTK. Inhibition of Jak-2 activity by a specific tyrosine kinase blocker, AG-490, selectively blocks leukaemic cell growthin vitro and in vivo by inducing programmed cell death, with no deleterious effect on normal haematopoiesis.

Structure and biogenesis of the cell walls of grasses

Abstract: The chemical structures of the primary cell walls of the grasses and their progenitors differ from those of all other flowering plant species. They vary in the complex glycans that interlace and cross-link the cellulose microfibrils to form a strong framework, in the nature of the gel matrix surrounding this framework, and in the types of aromatic substances and structural proteins that covalently cross-link the primary and secondary walls and lock cells into shape. This review focuses on the chemistry of the unique polysaccharides, aromatic substances, and proteins of the grasses and how these structural elements are synthesized and assembled into dynamic and functional cell walls. Despite wide differences in wall composition, the developmental physiology of grasses is similar to that of all flowering plants. Grass cells respond similarly to environmental cues and growth regulators, exhibit the same alterations in physical properties of the wall to allow cell growth, and possess similar patterns of wall biogenesis during the development of specific cell and tissue types. Possible unifying mechanisms of growth are suggested to explain how grasses perform the same wall functions as other plants but with different constituents and architecture.

Expression of galectin-3 modulates T-cell growth and apoptosis

Abstract: Galectin-3 is a member (if a large family of beta-galactoside-binding animal lectins. It has been shown that the expression of galectin-3 is upregulated in proliferating cells, suggesting a possible role for this lectin in regulation of cell growth. Previously, we have shown that T cells infected with human T-cell leukemia virus type I express high levels of galectin-3, in contrast to uninfected cells, which do not express detectable amounts of this protein. In this study, we examined growth properties of human leukemia T cells transfected with galectin-3 cDNA, and thus constitutively overexpressing this lectin. Transfectants expressing galectin-3 displayed higher growth rates than control transfectants, which do not express this lectin. Furthermore, galectin-3 expression in these cells confers resistance to apoptosis induced by anti-Fas antibody and staurosporine. Galectin-3 was found to have significant sequence similarity with Bcl-2, a well-characterized suppressor of apoptosis. In particular, the lectin contains the NWGR motif that is highly conserved among members of the Bcl-2 family and shown to be critical for the apoptosis-suppressing activity. We further demonstrated that galectin-3 interacts with Bc1-2 in a lactose-inhibitable manner. We conclude that galectin-3 is a regulator of cell growth and apoptosis and it may function through a cell death inhibition pathway that involves Bcl-2.

The microtubule-affecting drug paclitaxel has antiangiogenic activity.

Abstract: Endothelial cell migration is a critical event during angiogenesis, and inhibitors of cell motility can affect the angiogenic process. Paclitaxel (Taxol(R)), a microtubule-stabilizing antineoplastic cytotoxic drug, inhibits motility and invasiveness of several cell types. The aim of this study was to investigate the effect of paclitaxel on endothelial cell functions and on angiogenesis. In vivo, paclitaxel (20-28 mg/kg i.v.) significantly inhibited the angiogenic response induced by tumor cell supernatant embedded in a pellet of reconstituted basement membrane (Matrigel) injected s.c. into C57BL/6N mice. In vitro, paclitaxel inhibited endothelial cell proliferation, motility, invasiveness, and cord formation on Matrigel in a dose-dependent manner. The antiangiogenic activity of paclitaxel was not linked to its cytotoxicity, since inhibition of endothelial cell chemotaxis and invasiveness occurred at drug concentrations which did not affect endothelial cell proliferation. Another cytotoxic drug, cisplatin, that inhibited endothelial cell proliferation in vitro, did not affect angiogenesis in vivo. These data indicate that paclitaxel has a strong antiangiogenic activity, a property that might contribute to its antineoplastic activity in vivo.

A central role for Stat3 in IL‐6‐induced regulation of growth and differentiation in M1 leukemia cells.

Abstract: Interleukin-6 (IL-6) induces either differentiation or growth of a variety of cells. Little is known about the molecular basis of this cellular decision. The family of signal transducer and activator of transcription (Stat) proteins are involved in signaling through a variety of cytokine and growth factor receptors, although their biological roles have not been established. To address whether Stat proteins play roles in IL-6-induced growth or differentiation, we introduced two types of mutant Stat3 acting in a dominant-negative manner into M1 leukemic cells which respond to IL-6 with growth arrest and terminal differentiation. We show that dominant-negative forms of Stat3 inhibited both IL-6-induced growth arrest at G(0)/G1 and macrophage differentiation in the M1 transformants. Blocking of Stat activation resulted in inhibition of IL-6-induced repression of c-myb and c-myc. Furthermore, IL-6 enhanced the growth of M1 cells primarily through shortening the length of the G1 period when Stat3 was suppressed. Thus IL-6 generates both growth-enhancing signals and growth arrest- and differentiation-inducing signals at the same time. Stat3 may be a key molecule which determines the cellular decision from cell growth to differentiation in M1 cells.

Inhibition of focal adhesion kinase (FAK) signaling in focal adhesions decreases cell motility and proliferation.

Abstract: It has been proposed that the focal adhesion kinase (FAK) mediates focal adhesion formation through tyrosine phosphorylation during cell adhesion. We investigated the role of FAK in focal adhesion structure and function. Loading cells with a glutathione-S-transferase fusion protein (GST-Cterm) containing the FAK focal adhesion targeting sequence, but not the kinase domain, decreased the association of endogenous FAK with focal adhesions. This displacement of endogenous FAK in both BALB/c 3T3 cells and human umbilical vein endothelial cells loaded with GST-Cterm decreased focal adhesion phosphotyrosine content. Neither cell type, however, exhibited a reduction in focal adhesions after GST-Cterm loading. These results indicate that FAK mediates adhesion-associated tyrosine phosphorylation, but not the formation of focal adhesions. We then examined the effect of inhibiting FAK function on other adhesion-dependent cell behavior. Cells microinjected with GST-Cterm exhibited decreased migration. In addition, cells injected with GST-Cterm had decreased DNA synthesis compared with control-injected or noninjected cells. These findings suggest that FAK functions in the regulation of cell migration and cell proliferation.

Apoptosis and apc in colorectal tumorigenesis

Abstract: Tumors result from disruptions in the homeostatic mechanisms that regulate cell birth and cell death In colon cancer, one of the earliest manifestation of this imbalance is the formation of polyps, caused by somatic and inherited mutations of the adenomatous polyposis coli (APC) tumor suppressor gene in both humans and mice While the importance of APC in tumorigenesis is well documented, how it functions to prevent tumors remains a mystery Using a novel inducible expression system, we show that expression of APC in human colorectal cancer cells containing endogenous inactive APC alleles results in a substantial diminution of cell growth Further evaluation demonstrated that this was due to the induction of cell death through apoptosis These results suggest that apoptosis plays a role not only in advanced tumors but also at the very earliest stages of neoplasia

The Drosophila phosphoinositide 3-kinase Dp110 promotes cell growth.

Abstract: Phosphoinositide 3-kinases (PI3Ks) have been identified in an evolutionarily diverse range of organisms, including mammals, Drosophila, yeast, plants and Dictyostelium They are activated by a multitude of extracellular signals and implicated in mitogenesis, differentiation and cell survival, as well as in the control of the cytoskeleton and cell shape Here we describe the molecular and functional analysis of Drosophila p110 (Dp110) A full-length Dp110 cDNA was isolated and found to encode a protein homologous throughout its length to the class I mammalian PI3Ks p110alpha and p110beta Overexpression of Dp110 in wing or eye imaginal discs resulted in flies with enlarged wings or eyes respectively In contrast, overexpression of Dp110 containing a mutation predicted to result in the loss of catalytic activity resulted in smaller wings and eyes The alterations in wing size result from changes in both cell size and cell number, whereas in the eye only differences in cell size were detected These data imply a role for Dp110 in growth control during Drosophila development and have implications for the function of class I PI3Ks in other organisms

Identification of a novel p53 functional domain that is necessary for efficient growth suppression

Abstract: Activation of the p53 tumor suppressor protein has been demonstrated to block cell growth by inducing either a transient cell cycle arrest or programmed cell death (apoptosis). Although evidence exists linking p53’s function as an activator of transcription to its ability to effect cell cycle arrest, the role of this activity in the induction of apoptosis remains unclear. To gain insight into the molecular mechanisms underlying p53-mediated antiproliferative pathways, a study was initiated to explore the functions of a putative p53 signaling domain. This region of the human p53 protein is localized between amino acids 61 and 94 (out of 393) and is noteworthy in that it contains five repeats of the sequence PXXP (where P represents proline and X any amino acid). This motif has been shown to play a role in signal transduction via its SH3 domain binding activity. A p53 cDNA deletion mutant (ΔproAE), which lacks this entire proline-rich domain (deleted for amino acids 62–91), was created and characterized for a variety of p53 functions. The entire domain has been shown to be completely dispensable for transcriptional activation. On the other hand, this deletion of the p53 proline-rich domain impairs p53’s ability to suppress tumor cell growth in culture. Amino acid substitution mutations at residues 22 and 23 of p53 (eliminates transcriptional activity) also impair p53-mediated inhibition of cell growth in culture. Unlike wild-type p53, the ΔproAE mutant cDNA can be stably expressed in tumor derived cell lines with few immediate detrimental effects. These cells express physiologic levels of p53 protein that are induced normally in response to DNA damage, indicating that removal of the proline-rich domain does not disrupt p53’s upstream regulation by DNA damage. These data indicate that, in addition to the transcriptional activation domain, the p53 proline-rich domain plays a critical role in the transmission of antiproliferative signals downstream of the p53 protein and may link p53 to a direct signal transduction pathway.

Nitric oxide mediates mitogenic effect of VEGF on coronary venular endothelium

Abstract: Vascular endothelial growth factor (VEGF) is a secreted protein that is a specific growth factor for endothelial cells. We have recently demonstrated that nitric oxide (NO) donors and vasoactive peptides promoting NO-mediated vasorelaxation induce angiogenesis in vivo as well as endothelial cell growth and motility in vitro; in contrast, inhibitors of NO synthase suppress angiogenesis. In this study we investigated the role of NO in mediating the mitogenic effect of VEGF on cultured microvascular endothelium isolated from coronary postcapillary venules. VEGF induced a dose-dependent increase in cell proliferation and DNA synthesis. The role of NO was determined by monitoring proliferation or guanosine 3',5'-cyclic monophosphate (cGMP) levels in the presence and absence of NO synthase blockers. The proliferative effect evoked by VEGF was reduced by pretreatment of the cells with NO synthase inhibitors. Exposure of the cells to VEGF induced a significant increment in cGMP levels. This effect was potentiated by superoxide dismutase addition and was abolished by NO synthase inhibitors. VEGF stimulates proliferation of postcapillary endothelial cells through the production of NO and cGMP accumulation.

p150Ship, a signal transduction molecule with inositol polyphosphate-5-phosphatase activity.

Abstract: The production, survival, and function of monocytes and macrophages is regulated by the macrophage colony-stimulating factor (M-CSF or CSF-1) through its tyrosine kinase receptor Fms. Binding of M-CSF to Fms induces the tyrosine phosphorylation and association of a 150-kD protein with the phosphotyrosine-binding (PTB) domain of Shc. We have cloned p150 using a modified yeast two-hybrid screen. p150 contains one SH2 domain, two potential PTB-binding sites, an ATP/GTP-binding domain, several potential SH3-binding sites, and a domain with homology to inositol polyphosphate-5-phosphatases. p150 antibodies detect this protein in FDC-P1 myeloid cells, but the same protein is not detectable in fibroblasts. The antibodies immunoprecipitate a 150-kD protein from quiescent or M-CSF-stimulated FDC-P1 cells that hydrolyzes PtdIns(3,4,5)P3, to PtdIns(3,4)P2. This activity is observed in Shc immunoprecipitates only after M-CSF stimulation. Retroviral expression of p15O in FD-Fms cells results in strong inhibition of cell growth in M-CSF and a lesser inhibition in IL-3. Ectopic expression of p150 in fibroblasts does not inhibit growth. This novel protein, p150(ship) (SH2-containing inositol phosphatase), identifies a component of a new growth factor-receptor signaling pathway in hematopoietic cells.

Role of programmed (apoptotic) cell death during the progression and therapy for prostate cancer.

Abstract: Cells possess within their epigenetic repertoire the ability to undergo an active process of cellular suicide termed programmed (or apoptotic) cell death. This programmed cell death process involves an epigenetic reprogramming of the cell that results in an energy-dependent cascade of biochemical and morphologic changes (also termed apoptosis) within the cell, resulting in its death and elimination. Although the final steps (i.e., DNA and cellular fragmentation) are common to cells undergoing programmed cell death, the activation of this death process is initiated either by sufficient injury to the cell induced by various exogenous damaging agents (e.g., radiation, chemicals, viruses) or by changes in the levels of a series of endogenous signals (e.g., hormones and growth/survival factors). Within the prostate, androgens are capable of both stimulating proliferation as well as inhibiting the rate of the glandular epithelial cell death. Androgen withdrawal triggers the programmed cell death pathway in both normal prostate glandular epithelia and androgen-dependent prostate cancer cells. Androgen-independent prostate cancer cells do not initiate the programmed cell death pathway upon androgen ablation; however, they do retain the cellular machinery necessary to activate the programmed cell death cascade when sufficiently damaged by exogenous agents. In the normal prostate epithelium, cell proliferation is balanced by a equal rate of programmed cell death, such that neither involution nor overgrowth normal occurs. In prostatic cancer, however, this balance is lost, such that there is greater proliferation than death producing continuous net growth. Thus, an imbalance in programmed cell death must occur during prostatic cancer progression. The goal of effective therapy for prostatic cancer, therefore, is to correct this imbalance. Unfortunately, this has not been achieved and metastatic prostatic cancer is still a lethal disease for which no curative therapy is currently available. In order to develop such effective therapy, an understanding of the programmed death pathway, and what controls it, is critical. Thus, a review of the present state of knowledge concerning programmed cell death of normal and malignant prostatic cells will be presented.

Differentiation pathways and histogenetic aspects of normal and abnormal prostatic growth: a stem cell model.

Abstract: A stem cell model is presented for the organization of the prostatic epithelium that may explain normal and abnormal growth in the human prostate. This model is based on recent data indicating that: 1) The three basic cell types encountered in the prostatic epithelium--i.e., secretory luminal, basal, and endocrine paracrine (EP) cells--are linked in the precursor progeny relationship. 2) The proliferative compartment of the normal and hyperplastic epithelium is located in the basal cell layer. 3) The proliferative compartment of the prostatic epithelium is androgen-independent but contains andro-responsive target cells. 4) During the malignant transformation of the prostatic epithelium, the proliferative zone is inverted and shifts to luminal cell types. 5) Formation of neoplastic basement membrane (BM) material is crucial for the development of the invasive phenotype in prostate cancer. 6) The proliferative activities in prostate cancer are exclusively restricted to exocrine cell types, whereas endocrine differentiated tumor cells are postmitotic cells. 7) The majority of exocrine tumor cells are androgen-responsive in contrast to endocrine differentiated cell types that consistently lack the nuclear androgen receptor (AR). In this model, a small stem cell population located in the basal cell layer gives rise to all epithelial cell lineages encountered in the normal, hyperplastic, and neoplastic prostate. The differentiating process from basal cells to secretory luminal cells via intermediate phenotypes is induced by circulating androgens, and largely depends on the presence of androgen-responsive target cells in the basal cell layer. Accordingly, the abnormal growth of the secretory epithelium in benign prostate hyperplasia (BPH) may be related to an increase in the total number of androgen-responsive basal cells in the proliferative compartment. Prostate cancer derives from transformed stem cells located in the basal cell layer that acquire secretory luminal characteristics under androgenic stimulation. During tumor invasion, the malignant phenotypes adhere via specific receptors to newly formed BM-material, which, in turn, may facilitate their passage through the extracellular matrix. The occurrence of endocrine differentiation in prostate cancer reflects the pluripotency of its stem cells. The widespread absence of nuclear AR in endocrine differentiated tumor cells clearly indicates that this phenotype belongs to those cell clones in prostate cancer, that are initially androgen-independent and refractory to hormonal therapy. Accordingly, the progressive emergence of endocrine cell clones during tumor progression may represent one mechanism by which prostate cancer cells escape hormonal control.

A Common Mutant Epidermal Growth Factor Receptor Confers Enhanced Tumorigenicity on Human Glioblastoma Cells by Increasing Proliferation and Reducing Apoptosis

Abstract: Alterations of the EGFR gene occur frequently in human gliomas where the most common is an in-frame deletion of exons 2–7 from the extracellular domain, resulting in a truncated mutant receptor (ΔEGFR or de 2–7 EGFR). We previously demonstrated that introduction of ΔEGFR into human U87MG glioblastoma cells (U87MG.ΔEGFR) conferred remarkably enhanced tumorigenicity in vivo . Here, we show by cell-mixing experiments that the enhanced tumorigenicity conferred by ΔEGFR is attributable to a growth advantage intrinsic to cells expressing the mutant receptor. We analyzed the labeling index of the proliferation markers Ki-67 and bromodeoxyuridine and found that tumors derived from U87MG.ΔEGFR cells had significantly higher labeling indexes than those of tumors derived from U87MG cells that were either naive, expressed kinase-deficient mutants of ΔEGFR, or overexpressed exogenous wild-type EGFR. We also utilized terminal deoxynucleotidyl transferase-mediated nick end-labeling assays and showed that the apoptotic index of U87MG.ΔEGFR tumors was more than 4-fold lower than that of parental U87MG tumors. This decrease in cell death was inversely correlated with the expression level of Bcl-XL, a negative regulator of apoptosis, which was more than 3-fold higher in U87MG.ΔEGFR-derived tumors than in those derived from parental cells. Similar observations were obtained in vitro in serum-free conditions. These results suggest that ΔEGFR exerts its pronounced enhancement of glioblastoma tumorigenicity by stimulating proliferation and inhibiting apoptosis and that the effects are directly attributable to its constitutively active signal.

The Biochemical and Cellular Basis of Cell Proliferation Assays That Use Tetrazolium Salts

Abstract: The MTT assay is widely used in cell proliferation and cytotoxicity assays. Most cellular bioreduction of MTT is associated with enzymes of the endoplasmic reticulum and involves the reduced pyridine nucleotides NADH and, to a lesser extent, NADPH. Succinate is a weak electron donor for mitochondrial MTT reduction. Recently, new tetrazolium salt assays that use intermediate electron acceptors to facilitate reduction have been introduced. In this article, we compare the biochemical basis of reduction of MTT, XTT, and WST-1. Unlike MTT, XTT and WST-1 are efficiently reduced by NADH and NADPH in the absence of cells or enzymes, and their reduction involves superoxide. Cellular reduction of WST-1 occurs at the cell surface and also involves superoxide.

Developmental Control of Cell Cycle Regulators: A Fly's Perspective

Abstract: During early development in many species, maternally supplied gene products permit the cell cycle to run at maximum velocity, subdividing the fertilized egg into smaller and smaller cells. As development proceeds, zygotic controls are activated that first limit divisions to defined spatial and temporal domains, coordinating them with morphogenesis, and then halt proliferation altogether, to allow cell differentiation. Analysis of the regulation of cyclin-dependent kinases (Cdks) in Drosophila has provided insights into how this embryonic program of cell proliferation is controlled at the molecular level and how it is linked to developmental cues. Recent studies have also begun to reveal how cell proliferation is controlled during the second phase of Drosophila development, which occurs in imaginal tissues. In contrast to their embryonic progenitors, imaginal cells proliferate with a cycle that requires cell growth and is linked to patterning processes controlled by secreted cell signaling molecules. The functions of these signaling molecules appear to be nearly as conserved between vertebrates and invertebrates as the cell cycle control apparatus itself, suggesting that the mechanisms that coordinate growth, patterning, and cell proliferation in developing tissues have ancient origins.

Suppression of interleukin-3-induced gene expression by a C-terminal truncated Stat5: role of Stat5 in proliferation.

Abstract: Interleukin-3 (IL3) was shown recently to utilize the transcription factor Stat5, but the genes regulated by this pathway and the biological consequence of Stat5 activation remained to be determined. In order to study the role of Stat5 in IL3 signalling, we constructed a dominant-negative Stat5 protein by C-terminal truncation, and inducibly expressed it in an IL3-dependent cell line. The effect of dominant-negative Stat5 induction on expression of IL3 early response genes was examined, and expression of several genes, including cis, osm and pim-1 was inhibited profoundly. The expression of c-fos was also reduced, but to a lesser extent. While activated Ras alone (though not Stat5 alone) could induce c-fos, maximal expression required the action of both Ras and Stat5. Interestingly, although the membrane-proximal region of the IL3 receptor beta-chain is responsible for both Jak2-Stat5 activation and c-myc induction, c-myc levels were not affected by the dominant-negative Stat5. Thus, the signals directed by this membrane-proximal domain, which is essential for transducing a DNA synthesis signal, can be separated further into Stat5 or c-myc pathways. The net effect of dominant-negative Stat5 expression was partial inhibition of IL3-dependent growth. This provides the first direct evidence that Stat5 is involved in regulation of cell proliferation.

Hepatitis C virus core protein cooperates with ras and transforms primary rat embryo fibroblasts to tumorigenic phenotype.

Abstract: We have previously demonstrated that hepatitis C virus (HCV) core protein regulates cellular protooncogenes at the transcriptional level; this observation implicates core protein in the alteration of normal hepatocyte growth. In the present study, the transforming potential of the HCV core gene was investigated by using primary rat embryo fibroblast (REF) cells which were transfected with or without cooperative oncogenes. Integration of the HCV core gene resulted in expression of the viral protein in REF stable transformants. REF cells cotransfected with HCV core and H-ras genes became transformed and exhibited rapid proliferation, anchor-independent growth, and tumor formation in athymic nude mice. Results from these studies suggest that the core protein plays an important role in the regulation of HCV-infected cell growth and in the transformation to tumorigenic phenotype. These observations suggest a possible mechanism for this viral protein in the pathogenesis of hepatocellular carcinoma in HCV-infected humans.

CCAAT/enhancer-binding protein alpha (C/EBP alpha) inhibits cell proliferation through the p21 (WAF-1/CIP-1/SDI-1) protein.

Abstract: C/EBPc~ has a role in growth arrest and differentiation of mouse preadipocytes. To study the mechanism of C/EBP,v-induced growth arrest, we developed a cell line, HT1, that contained the human C/EBP~ gene under Lac repressor control. IPTG-induced C/EBPc~ caused inhibition of cell proliferation and DNA synthesis as measured by colony growth assays, cell counting, and BrdU uptake. A number of proteins that are known to be involved in the regulation of the cell cycle, such as cyclin-dependent kinase (CDK) 2 and CDK4, proliferating cell nuclear antigen (PCNA), p53, c-fos, and the CDK inhibitor p16 and p27 were investigated by Western analysis. No change in their expression was observed. However, the p21 (WAF-1/CIP-1 / SDI-1) protein was significantly elevated in growth-arrested HT1 cells. Elevation of p21/SDI-1 mRNA (threefold) and activation of the p21/SDI-1 promoter by C/EBPot did not account for the 12- to 20-fold increase in p21/SDI-1 protein. Protein synthesis inhibition by cycloheximide (CHX) treatment indicated that the half-life of p21/SDI-1 in dividing HT1 cells was -30 min. However, in C/EBP,~ growth-arrested cells, the level of the p21/SDI-1 did not change for >80 rain after CHX addition. Our studies demonstrate that C/EBPa activates p21/SDI-1 by increasing p21/SDI-1 gene expression and by post-translational stabilization of p21/SDI-1 protein. Furthermore, induction of p21/SDI-1 is responsible for the ability of C/EBP,v to inhibit proliferation because transcription of antisense p21/SDI-1 mRNA eliminated growth inhibition by C/EBPa.