Showing papers on "Cell growth published in 1988"

Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line.

Abstract: In contrast to mouse epidermal cells, human skin keratinocytes are rather resistant to transformation in vitro. Immortalization has been achieved by SV40 but has resulted in cell lines with altered differentiation. We have established a spontaneously transformed human epithelial cell line from adult skin, which maintains full epidermal differentiation capacity. This HaCaT cell line is obviously immortal (greater than 140 passages), has a transformed phenotype in vitro (clonogenic on plastic and in agar) but remains nontumorigenic. Despite the altered and unlimited growth potential, HaCaT cells, similar to normal keratinocytes, reform an orderly structured and differentiated epidermal tissue when transplanted onto nude mice. Differentiation-specific keratins (Nos. 1 and 10) and other markers (involucrin and filaggrin) are expressed and regularly located. Thus, HaCaT is the first permanent epithelial cell line from adult human skin that exhibits normal differentiation and provides a promising tool for studying regulation of keratinization in human cells. On karyotyping this line is aneuploid (initially hypodiploid) with unique stable marker chromosomes indicating monoclonal origin. The identity of the HaCaT line with the tissue of origin was proven by DNA fingerprinting using hypervariable minisatellite probes. This is the first demonstration that the DNA fingerprint pattern is unaffected by long-term cultivation, transformation, and multiple chromosomal alterations, thereby offering a unique possibility for unequivocal identification of human cell lines. The characteristics of the HaCaT cell line clearly document that spontaneous transformation of human adult keratinocytes can occur in vitro and is associated with sequential chromosomal alterations, though not obligatorily linked to major defects in differentiation.

Angiotensin II induces hypertrophy, not hyperplasia, of cultured rat aortic smooth muscle cells.

Abstract: We have explored the hypothesis that contractile agonists are important regulators of smooth muscle cell growth by examining the effects of one potent contractile agonist, angiotensin II (AII), on both cell proliferation and cellular hypertrophy. AII neither stimulated proliferation of cells made quiescent in a defined serum-free media nor augmented cell proliferation induced by serum or platelet-derived growth factor. However, AII did induce cellular hypertrophy of postconfluent quiescent cultures following 4 days of treatment, increasing smooth muscle cell protein content by 20% as compared with vehicle-treated controls. AII-induced hypertrophy was maximal at 1 microM, had an ED50 of 5 nM, and was blocked by the specific AII receptor antagonist Sar1,Ile8 AII. The cellular hypertrophy was due to an increase in protein synthesis, which was elevated within 6-9 hours following AII treatment, while no changes in protein degradation were apparent. AII was even more effective in inducing hypertrophy of subconfluent cultures, causing a 38% increase in protein content after 4 days of treatment (1 microM) and showing a maximal response at concentrations as low as 0.1 nM. Interestingly, in subconfluent cultures, AII treatment (1 microM, 4 days) was associated with a 50% increase in the fraction of cells with 4C DNA content with the virtual absence of cells in S-phase of the cell cycle, consistent with either arrest of cells in the G2 phase of the cell cycle or development of tetraploidy.(ABSTRACT TRUNCATED AT 250 WORDS)

Heparan sulphate bound growth factors: a mechanism for stromal cell mediated haemopoiesis.

Abstract: The proliferation and development of haemopoietic stem cells takes place in close association with marrow stromal cells1,2. This intimate cell contact presumably enables the stem cells and their progeny to respond to stimuli present on the stromal cell surface. While the nature of these stimuli has not been determined, it is likely that growth factors play some role. Recently, it was demonstrated that the natural and the recombinant haemopoietic growth factor, granulocyte/macrophage colony stimulating factor (GM-CSF), could be adsorbed out of solution by an extract of human marrow stromal extracellular matrix (ECM) with retention of biological activity3. However, the precise ECM molecules involved were not identified. Here, we clearly demonstrate that the major sulphated glycosaminoglycan of mouse marrow stroma, heparan sulphate4, possesses the ability to adsorb both GM-CSF and the multilineage haemopoietic growth factor, Interleukin 3 (IL-3). Furthermore, these growth factors, once bound, can be presented in the biologically active form to haemopoietic cells.

Peptide growth factors are multifunctional

Abstract: The actions of many peptide growth factors include both stimulation and inhibition of cell proliferation, as well as effects unrelated to the control of cell growth. One peptide can have both stimulatory and inhibitory activity in a single cell, depending on the context of the other signal molecules present.

Stimulation of B-cell progenitors by cloned murine interleukin-7.

Abstract: The events involved in the commitment and development of lymphoid lineage cells are poorly understood. We have used a recently described long-term culture system1 to establish a bioassay that can detect a novel growth factor capable of stimulating the proliferation of lymphoid progenitors2. Using direct expression in mammalian cells we have isolated a complementary DNA clone encoding this novel haematopoietic growth factor, designated interleukin-7.

Inhibition of NIH 3T3 cell proliferation by a mutant ras protein with preferential affinity for GDP.

Abstract: Substitution of asparagine for serine at position 17 decreased the affinity of rasH p21 for GTP 20- to 40-fold without significantly affecting its affinity for GDP. Transfection of NIH 3T3 cells with a mammalian expression vector containing the Asn-17 rasH gene and a Neor gene under the control of the same promoter yielded only a small fraction of the expected number of G418-resistant colonies, indicating that expression of Asn-17 p21 inhibited cell proliferation. The inhibitory effect of Asn-17 p21 required its localization to the plasma membrane and was reversed by coexpression of an activated ras gene, indicating that the mutant p21 blocked the endogenous ras function required for NIH 3T3 cell proliferation. NIH 3T3 cells transformed by v-mos and v-raf, but not v-src, were resistant to inhibition by Asn-17 p21, indicating that the requirement for normal ras function can be bypassed by these cytoplasmic oncogenes. The Asn-17 mutant represents a novel reagent for the study of ras function by virtue of its ability to inhibit cellular ras activity in vivo. Since this phenotype is likely associated with the preferential affinity of the mutant protein for GDP, analogous mutations might also yield inhibitors of other proteins whose activities are regulated by guanine nucleotide binding.

Proliferation and differentiation of rat neuroepithelial precursor cells in vivo

Abstract: Important features of adult neuronal number, location, and type are a consequence of early embryonic events that occur before neurons have differentiated. We have measured cell number during embryogenesis of the rat CNS. Markers that are expressed in the proliferating neuronal precursor are required to study the mechanisms controlling their proliferation and differentiation. By applying immunohistochemistry, fluorescence-activated cell sorting, and 3H-thymidine auto-radiography to dissociated rat CNS cells, we show that the monoclonal antibody Rat 401 recognizes a cell population with proliferative, temporal, and quantitative features expected of neuronal precursors.

Induction of proto-oncogene JUN /AP-1 by serum and TPA

Abstract: The response of a cell to mitogens and differentiation agents involves the transcriptional induction of several cellular genes. Prominent among these so-called 'immediate early' or 'competence' genes are the nuclear oncogenes fos and mycl–7. Although the precise function of these early response genes in growth control is not understood, it is likely that many of them are involved in the transition from G0 to G1 in the cell cycle. The findings that the products of nuclear proto-oncogenes jun and erbA are transcriptional factors supports the notion of the role of the nuclear oncoproteins in the regulation of gene expression8–11. Recently, it has been reported that the FOS protein is associated in transcriptional complexes with the product of the jun oncogene, the transcription factor AP-1 (refs 12–15). As the fos gene is induced in response to mitogens during initiation of cell growth, we investigated whether expression of the nuclear transcription factor AP-1 is also inducible. We report that mouse c-jun gene transcription is rapidly induced by serum and phorbol-ester 12-o-tetradecanoyl phorbol 13-acetate (TPA). Furthermore, induction is transient and the mRNA is superinduced by inhibitors of protein synthesis.

Transcriptional activation of c-jun during the G0/G1 transition in mouse fibroblasts

Abstract: Before quiescent cells can respond to mitogens and progress through the G1 phase of cell growth, new messenger RNA synthesis is required1 The G1 phase seems to be a critical point of control in the cell cycle, where normal cells deprived of growth factors halt cycling while transformed cells do not, suggesting that regulatory genes, uncontrolled in the neoplastic phenotype, are expressed during the G0 to G1 transition Some of these may code for nuclear proteins that participate in the transactivation of genes required for the progression through G1 The observed changes in expression of the proto-oncogenes c-fos and c-myc, following stimulation of fibroblasts with growth factors2–7, support this notion as recent evidence suggests that c-FOS and c-MYC proteins can function as transactivating factors8–12 Moreover, the rapid induction of several genes in fibroblasts coding for putative transacting factors during the G0 to G1 transition has been recently reported13–16 Here we present the nucleotide sequence of a mouse cDNA clone coding for a 334 residue protein which shows 80% similarity with v-JUN17 and more than 98% similarity with the human c-JUN sequence18,19 We have demonstrated that in quiescent fibroblasts c-jun transcription is rapidly induced during the G0 to G1 transition

An oligomer complementary to c-myc mRNA inhibits proliferation of HL-60 promyelocytic cells and induces differentiation

Abstract: To study the role of a nuclear proto-oncogene in the regulation of cell growth and differentiation, we inhibited HL-60 c-myc expression with a complementary antisense oligomer. This oligomer was stable in culture and entered cells, forming an intracellular duplex. Incubation of cells with the anti-myc oligomer decreased the steady-state levels of c-myc protein by 50 to 80%, whereas a control oligomer did not significantly affect the c-myc protein concentration. Direct inhibition of c-myc expression with the anti-myc oligomer was associated with a decreased cell growth rate and an induction of myeloid differentiation. Related antisense oligomers with 2- to 12-base-pair mismatches with c-myc mRNA did not influence HL-60 cells. Thus, the effects of the antisense oligomer exhibited sequence specificity, and furthermore, these effects could be reversed by hybridization competition with another complementary oligomer. Antisense inhibition of a nuclear proto-oncogene apparently bypasses cell surface events in affecting cell proliferation and differentiation.

Expression of transforming growth factor α and its messenger ribonucleic acid in human breast cancer: its regulation by estrogen and its possible functional significance

Abstract: We have studied the estrogenic regulation and the potential autocrine role of transforming growth factor alpha (TGF alpha) in the human breast cancer cell line MCF-7. A biologically active apparent mol wt 30 k TGF alpha was identified by gel filtration chromatography in medium conditioned by MCF-7 breast cancer cells. We previously reported induction of TGF alpha levels in medium by 17 beta-estradiol. We now report correlated increases in TGF alpha mRNA, by Northern and slot blot analysis, after estrogen treatment of MCF-7 cells in vitro. In vivo experiments confirmed these data: estrogen withdrawal from MCF-7 tumor-bearing nude mice resulted in a decline in tumor size and TGF alpha mRNA levels. To explore the functional significance of TGF alpha in MCF-7 cells, anti-TGF alpha antibody was added to MCF-7 soft agar cloning assays. Inhibition of MCF-7 growth resulted, supporting an autocrine role for TGF alpha. Further experiments using an anti-EGF receptor antibody expanded this data, demonstrating inhibition of estrogen-stimulated monolayer MCF-7 cell growth. Examining the generality of TGF alpha expression, 4.8 kilobase TGF alpha mRNAs were seen in three other human breast cancer cell lines, MDA-MB-231, ZR 75B, and T47D. Expression of TGF alpha mRNA was detected in 70% of estrogen receptor positive and negative primary human breast tumors from 40 patients when examined by slot blot and Northern analysis. Thus, we have demonstrated broad expression of TGF alpha in human breast cancer, its hormonal regulation in an estrogen-responsive cell line, and its possible functional significance in MCF-7 cell growth.

Effects of the pineal hormone melatonin on the proliferation and morphological characteristics of human breast cancer cells (MCF-7) in culture.

Abstract: Since melatonin, the major hormone of the pineal gland, has been shown to inhibit the growth of mammary tumors in animal models of human breast cancer, we examined the hypothesis that this indoleamine has the potential to inhibit breast cancer growth by directly inhibiting cell proliferation as exemplified by the growth of the estrogen-responsive human breast cancer cell line MCF-7 in culture. Concentrations of melatonin (10(-9) M; 10(-11) M), corresponding to the physiological levels present in human blood during the evening hours, significantly inhibited (P less than 0.001) cell proliferation by as much as 60% to 78% as measured by either DNA content or hemocytometer cell counts. Melatonin's inhibitory effect was reversible since the logarithmic growth of MCF-7 cells was restored after melatonin-containing medium was replaced with fresh medium lacking melatonin. Not only was the inhibitory effect of melatonin absent at either pharmacological (10(-7) M; 10(-5) M) or subphysiological (10(-15) M; 10(-13) M) concentrations, but melatonin also failed to inhibit the proliferation of either human foreskin fibroblasts or the estrogen receptor-positive human endometrial cancer cell line RL95-2. Both transmission and scanning electron microscopy revealed several morphological changes that correlated with melatonin's inhibition of cell growth. After just 4 days of exposure to melatonin, MCF-7 cells exhibited reduced numbers of surface microvilli, nuclear swelling, cytoplasmic and ribosomal shedding, disruption of mitochondrial cristae, vesiculation of the smooth endoplasmic reticulum, and an increase in the numbers of autophagic vacuoles. These results support the hypothesis that melatonin, at physiological concentrations, exerts a direct but reversible, antiproliferative effect on MCF-7 cell growth in culture. This antiproliferative effect is associated with striking changes in the ultrastructural features of these cells suggestive of a sublethal but reversible cellular injury.

Functional discrimination between interleukin 6 and interleukin 1

Abstract: In this study we have investigated the specificity of bioassays in which interleukin (IL)1 and/or IL6 are active. The thymocyte assay cannot be used to discriminate between IL1 and IL6; both monokines are active in this assay. Moreover the detection limit for both IL1 and IL6 is around 100 pg/ml. IL6 activity can be measured with a murine hybridoma cell line (B9). The detection limit for human as well as murine IL6 is about 0.5 pg/ml. The assay is specific for IL 6 and is not influenced by a variety of other cytokines except for murine IL4 which shows some activity in this assay. IL1 can be measured specifically with D10 cells. The detection limit for IL1α and IL1β is around 1 pg/ml whereas IL6 is not active in this assay at all. Upon stimulation by IL1 and/or IL2 D10 cells produce IL6. However, this IL6 does not seem to be involved in the proliferation of these cells.

Epstein-Barr virus latent infection membrane protein alters the human B-lymphocyte phenotype: deletion of the amino terminus abolishes activity.

Abstract: A latent infection membrane protein (LMP) encoded by the Epstein-Barr virus (EBV) genome in latently infected, growth-transformed lymphocytes alters the phenotype of a human EBV-negative B-lymphoma cell line (Louckes) when introduced by gene transfer. These LMP-expressing cells exhibit increased homotypic adhesion due to increased expression of the adhesion molecules LFA-1 and ICAM-1. Increased homotypic adhesion could foster B-cell growth by facilitating autocrine growth factor effects. LFA-3 expression is also induced. The induction of LFA-3 and ICAM-1 results in increased heterotypic adhesion to T lymphocytes. This could result in more effective T-cell immune surveillance. Since LMP is expressed in EBV-transformed lymphocytes and has been demonstrated to transform rodent fibroblasts in vitro, a wide range of possible effects on B-lymphoma cell growth were assayed. In the Louckes B-lymphoma cell line, EBV LMP causes increased cell size, acid production, plasma membrane ruffling, and villous projections. Although cell proliferation rate was not greatly affected, the steady-state intracellular free calcium level, transforming growth factor beta responsiveness, and expression of the lymphocyte activation markers (CD23 and transferrin receptor) were increased. Thus, LMP appears to be a mediator of EBV effects on B-cell transformation. In transfected lymphoma cells, LMP localizes to patches at the cell periphery and associates with the cytoskeleton as it does in EBV-transformed B lymphocytes or in rodent fibroblasts. A partially deleted form of LMP (D1LMP) does not aggregate in patches or associate with the cytoskeleton and had little effect on B-cell growth. Thus, cytoskeletal association may be integral to LMP activity.

Human promyelocytic leukemia HL-60 cell proliferation and c-myc protein expression are inhibited by an antisense pentadecadeoxynucleotide targeted against c-myc mRNA

Abstract: The human promyelocytic leukemia cell line HL-60 overexpresses the c-myc protooncogene. A calculated secondary structure for c-myc mRNA placed the initiation codon in a bulge of a weakly base-paired region. Treatment of HL-60 cells with 5' d(AACGTTGAGGGGCAT) 3', complementary to the initiation codon and the next four codons of c-myc mRNA, inhibited c-myc protein expression in a dose-dependent manner. However, treatment of HL-60 cells with 5' d(TTGGGATAACACTTA) 3', complementary to nucleotides 17-31 of vesicular stomatitis virus matrix protein mRNA, displayed no such effects. These results agree with analogous studies of normal human T lymphocytes [Heikkila, R., Schwab, G., Wickstrom, E., Loke, S. L., Pluznik, D. H., Watt, R. & Neckers, L. M. (1987) Nature (London) 328, 445-449], except that only one-third as much oligomer was needed for a comparable effect. Proliferation of HL-60 cells in culture was inhibited in a sequence-specific, dose-dependent manner by the c-myc-complementary oligomer, but neither the oligomer complementary to vesicular stomatitis virus matrix protein mRNA nor 5' d(CATTTCTTGCTCTCC) 3', complementary to nucleotides 5399-5413 of human immunodeficiency virus tat gene mRNA, inhibited proliferation. It thus appears that antisense oligodeoxynucleotides added to myc-transformed cells via culture medium are capable of eliciting sequence-specific, dose-dependent inhibition of c-myc protein expression and cell proliferation.

Transforming growth factor-beta is a potent immunosuppressive agent that inhibits IL-1-dependent lymphocyte proliferation.

Abstract: Transforming growth factor-beta (TGF-beta), a product of neoplastic and hemopoietic cells, is a bifunctional regulator of the immune response. At femtomolar concentrations, TGF-beta stimulates monocyte migration, and picomolar quantities induce synthesis of monocyte growth factors, including IL-1, that may promote tissue repair by regulating fibrosis and angiogenesis. Paradoxically, TGF-beta at picomolar concentrations also blocks the ability of IL-1 to stimulate lymphocyte proliferation. At 0.01 to 1.0 ng/ml, TGF-beta 1 and its homologue, TGF-beta 2, suppress the IL-1-dependent murine thymocyte proliferation assay. TGF-beta also inhibits human peripheral blood T lymphocyte mitogenesis. Inhibition of cell division appears to occur after activation of the lymphocytes inasmuch as neither gene expression nor translation of IL-2R is suppressed. Furthermore, TGF-beta does not block synthesis of IL-2. Therefore, TGF-beta 1 and TGF-beta 2 likely act at a site distal to IL-1 to block lymphocyte DNA synthesis. These findings suggest that TGF-beta secreted in an inflammatory site may be beneficial in diminishing lymphocyte function while promoting fibrosis and tissue repair. However, TGF-beta generated by neoplastic tissues may provide a mechanism for unrestricted tumor cell growth through its selective immunosuppressive effects.

Insulin-like growth factor I promotes cell proliferation and oligodendroglial commitment in rat glial progenitor cells developing in vitro.

Abstract: We investigated the mechanisms by which insulin-like growth factor I (IGF-I) acts to increase the number of oligodendrocytes that develop in cultures of cells explanted from perinatal rat cerebrum. Fluorescence-activated cell sorting was used to isolate bipotential A2B5-positive oligodendrocyte-type 2 astrocyte (O-2A) progenitor cells, which were then inoculated as single cells into microculture wells containing feeder layers of X-irradiated type 1 astrocytes. Addition of 100 ng/ml IGF-I to the culture medium increased the growth rate and the ultimate size reached by the resulting clones during the 18-day experimental period. Moreover, 75-80% of the cells in the IGF-I-treated clones differentiated into galactocerebroside (GC)-positive oligodendrocytes, whereas only 25-30% became oligodendrocytes in the absence of IGF-I. IGF-I did not increase the number of type 2 astrocytes that developed in the clones. IGF-I appeared to have the greatest effect on growth and differentiation at a stage when the majority of the cells in the clones were at an intermediate stage of development, characterized by the expression of A2B5 and O4 glycolipid antigens but not GC. Analysis of the effects of IGF-I on O4-positive, GC-negative intermediate precursor cells revealed a two to fivefold increase in the number of cells that incorporated 3H-thymidine into their DNA during a 5-h pulse. Moreover, IGF-I increased the number of cell sorter-purified O4-positive cells that developed into oligodendrocytes 4-8 days later. Therefore, IGF-I acts in two different ways to promote oligodendrocyte development: It promotes proliferation of precursor cells in the O-2A lineage, and it induces precursors to become committed to develop into oligodendrocytes.

RH 5849, a nonsteroidal ecdysone agonist: effects on a Drosophila cell line

Abstract: The steroid molting hormone 20-hydroxyecdysone is the physiological inducer of molting and metamorphosis in insects. In ecdysone-sensitive Drosophila Kc cells, the insecticide RH 5849 (1,2-dibenzoyl-1-tert-butylhydrazine) mimics the action of 20-hydroxyecdysone by causing the formation of processes, an inhibition of cell proliferation, and induction of acetylcholinesterase. RH 5849 also competes with [3H]ponasterone A for high-affinity ecdysone receptor sites from Kc cell extracts. Resistant cell populations selected by growth in the continued presence of either RH 5849 or 20-hydroxyecdysone are insensitive to both compounds and exhibit a decreased titer of measurable ecdysone receptors. Although it is less potent than 20-hydroxyecdysone in both whole-cell and cell-free receptor assays, RH 5849 is the first nonsteroidal ecdysone agonist.

Basic fibroblast growth factor fused to a signal peptide transforms cells.

Abstract: Basic fibroblast growth factor (bFGF) is a potent growth and angiogenic factor that is found in abundance in tissues such as brain, hypothalamus, kidney and cartilage1,2. Despite this copious production of bFGF, most of these tissues are not undergoing either active growth or angiogenesis, suggesting that bFGF activity must be regulated so as to prevent autostimulation of cell growth. In cultured cells, bFGF is associated mainly with cells and basement membranes and is not released into the medium3,4. Prevention of release could be a mechanism for regulation of bFGF activity and may be a consequence of the apparent absence of a secretory-signal sequence in the bFGF protein5. Here we investigate whether this regulation can be overridden through the forced secretion of bFGF. Such secretion might provide the bFGF access to its receptor and in turn lead to autocrine transformation of the cell. We report that bFGF, as specified by a recombinant plasmid, is itself unable to induce such transformation, but acquires this ability after fusion with a secretory-signal sequence. The resulting transformants undergo unusual morphological alteration and display tumorigenicity.

Transforming growth factor-beta-induced growth inhibition and cellular hypertrophy in cultured vascular smooth muscle cells.

Abstract: We have explored the hypothesis that hypertrophy of vascular smooth muscle cells may be regulated, in part, by growth inhibitory factors that alter the pattern of the growth response to serum mitogens by characterizing the effects of the potent growth inhibitor, transforming growth factor-beta (TGF-beta), on both hyperplastic and hypertrophic growth of cultured rat aortic smooth muscle cells. TGF-beta inhibited serum-induced proliferation of rat aortic smooth muscle cells (ED50 = 2 pM); this is consistent with previously reported observations in bovine aortic smooth muscle cells (Assoian et al. 1982. J. Biol. Chem. 258:7155-7160). Growth inhibition was due in part to a greater than twofold increase in the cell cycle transit time in cells that continued to proliferate in the presence of TGF-beta. TGF-beta concurrently induced cellular hypertrophy as assessed by flow cytometric analysis of cellular protein content (47% increase) and forward angle light scatter (32-50% increase), an index of cell size. In addition to being time and concentration dependent, this hypertrophy was reversible. Simultaneous flow cytometric evaluation of forward angle light scatter and cellular DNA content demonstrated that TGF-beta-induced hypertrophy was not dependent on withdrawal of cells from the cell cycle nor was it dependent on growth arrest of cells at a particular point in the cell cycle in that both cycling cells in the G2 phase of the cell cycle and those in G1 were hypertrophied with respect to the corresponding cells in vehicle-treated controls. Chronic treatment with TGF-beta (100 pM, 9 d) was associated with accumulation of cells in the G2 phase of the cell cycle in the virtual absence of cells in S phase, whereas subsequent removal of TGF-beta from these cultures was associated with the appearance of a significant fraction of cycling cells with greater than 4c DNA content, consistent with development of tetraploidy. Results of these studies support a role for TGF-beta in the control of smooth muscle cell growth and suggest that at least one mechanism whereby hypertrophy and hyperploidy may occur in this, as well as other cell types, is by alterations in the response to serum mitogens by potent growth inhibitors such as TGF-beta.

Correlation of Modified Human Papilloma Virus Early Gene Expression with Altered Growth Properties in C4-1 Cervical Carcinoma Cells

Abstract: In cervical carcinomas and cell lines derived from these tumors the DNA of specific types of human papilloma viruses (HPVs) is integrated into the host cell genome. The two viral open reading frames E6 and E7 are consistently transcribed in tumors and cell lines, and the respective proteins were detected in cells cultured in vitro . As shown here, modulation of HPV 18 E6 and E7 gene expression in C4-1 cervical carcinoma cells is accompanied by an altered cell growth. HPV 18 E6 and E7 expression can be enhanced by glucocorticoid treatment of C4-1 cells, and an increased cell proliferation is observed. In contrast, after introduction of complementary RNA to the HPV 18 E6 and E7 open reading frames, their expression is inhibited, and decreased cell growth is observed. These results support the hypothesis that expression of HPV E6 and E7 open reading frames is directly involved in growth regulation of cervical carcinoma cells.

Enforced expression of the c-myc oncogene inhibits cell differentiation by precluding entry into a distinct predifferentiation state in G0/G1.

Abstract: A broad base of data has implicated a role for the c-myc proto-oncogene in the control of the cell cycle and cell differentiation. To further define the role of myc in these processes, I examined the effect of enforced myc expression on several events that are thought to be important steps leading to the terminally differentiated state: (i) the ability to arrest growth in G0/G1, (ii) the ability to replicate the genome upon initiation of the differentiation program, and (iii) the ability to lose responsiveness to mitogens and withdraw from the cell cycle. 3T3-L1 preadipocyte cell lines expressing various levels of myc mRNA were established by transfection with a recombinant myc gene under the transcriptional control of the Rous sarcoma virus (RSV) promoter. Cells that expressed high constitutive levels of pRSVmyc mRNA arrested in G0/G1 at densities similar to those of normal cells at confluence. Upon initiation of the differentiation program, such cells traversed the cell cycle with kinetics similar to those of normal cells and subsequently arrested in G0/G1. Thus, enforced expression of myc had no effect on the ability of cells to arrest growth in G0/G1 or to replicate the genome upon initiation of the differentiation program. Cells were then tested for their ability to reenter the cell cycle upon exposure to high concentrations of serum and for their capacity to differentiate. In contrast to normal cells, cells expressing high constitutive levels of myc RNA reentered the cell cycle when challenged with 30% serum and failed to terminally differentiate. The block to differentiation could be reversed by high expression of myc antisense RNA, showing that the induced block was specifically due to enforced expression of pRSVmyc. These findings indicate that 3T3-L1 preadipocytes enter a specific state in G0/G1 after treatment with differentiation inducers, into which cells expressing high constitutive levels of myc RNA are precluded from entering. I propose that myc acts as a molecular switch and directs cells to a pathway that can lead to continued proliferation or to terminal differentiation.

Molecular events in the processing of recombinant type 1 pre-pro-transforming growth factor beta to the mature polypeptide.

Abstract: Recently, the simian type 1 transforming growth factor beta (TGF-beta 1) cDNA was expressed at high levels in Chinese hamster ovary (CHO) cells by dihydrofolate reductase-induced gene amplification (L.E. Gentry, N.R. Webb, G.J. Lim, A.M. Brunner, J.E. Ranchalis, D.R. Twardzik, M.N. Lioubin, H. Marquardt, and A.F. Purchio, Mol. Cell. Biol. 7:3418-3427, 1987). We have now purified and characterized the recombinant proteins released by these cells. Analyses of the precursor proteins by amino acid sequencing identified potentially important proteolytic processing sites. Signal peptide cleavage occurs at the Gly-29-Leu-30 peptide bond of pre-pro-TGF-beta 1, yielding pro-TGF-beta 1 (30 to 390). In addition, proteolytic processing of the precursor to yield mature TGF-beta 1 occurs at the dibasic cleavage site immediately preceding Ala-279, indicating that CHO cells possess the appropriate processing enzyme. Greater than 95% of the biological activity detected in the conditioned medium of the CHO transfectant was due to mature, properly processed growth factor. Highly purified recombinant TGF-beta 1 had the same specific biological activity as natural TGF-beta 1. The concentration of TGF-beta 1 required for half-maximal inhibition of Mv1Lu mink lung epithelial cell growth was approximately 1 to 2 pM. Purified precursor inhibited mink lung cell proliferation at 50 to 60 pM concentrations. The purified precursor preparation was shown to consist of pro-TGF-beta 1 (30 to 390), the pro region of the precursor (30 to 278), and mature TGF-beta 1 (279 to 390) interlinked by at least one disulfide bond with the pro portion of the precursor. These recombinant forms of TGF-beta1 should prove useful for further structural and functional studies.

Limiting dilution analysis of proliferative responses in human lymphocyte populations defined by the monoclonal antibody UCHL1: implications for differential CD45 expression in T cell memory formation.

Abstract: Using limiting dilution analysis, we investigated proliferative responses of UCHL1+ and UCHL1− T cell populations to compare the precursor cell frequencies following recall and alloantigen stimulation, and the complexity of cellular interactions within UCHL1+ and UCHL1− populations. We find high frequencies of recall antigen responses among UCHL l+, but not UCHL1− T cells. In contrast, both populations contain similar frequencies of alloantigen responsive cells. Our results are consistent with single-hit kinetics in recall as well as alloantigen responses and show no complex cellular interactions within the responding populations. In conclusion, the difference between the recall antigen response of UCHL 1+ and UCHL1− cells observed in conventional proliferation assays is most probably due to a high frequency of recall antigen-responsive UCHL1+ cells and not to suppressive phenomena in the UCHL1−population. These data suggest that memory T cells are largely of the UCHL 1+ phenotype. The relation of post-thymic T cell maturation and differential CD45 expression is briefly discussed.

Monocyte-derived human B-cell growth factor identified as interferon-beta 2 (BSF-2, IL-6).

Abstract: Soluble products of either Epstein-Barr virus (EBV)-infected B cells or activated monocytes promote the proliferation of EBV-infected B cells and permit their growth at low cell densities. This suggests that growth factors are important for B-cell immortalization by EBV. In this study, a monocyte-derived factor that promotes the growth of EBV-infected b cells was purified and identified as interferon-beta 2 (IFN-beta 2), which is also known as 26-kilodalton protein, B-cell differentiation factor (BSF-2), and interleukin-6 (IL-6). The purified protein has a specific activity of approximately 4 X 10(7) units per milligram of protein in assays of B-cell growth. Thus, IFN-beta 2/BSF-2 is a B-cell growth factor that promotes the proliferation of human B cells infected with EBV.