Human immunodeficiency virus 1 envelope-initiated G2-phase programmed cell death.

Despite intensive investigation, no clearly defined mechanism explaining human immunodeficiency virus (HIV)-induced cell killing has emerged. HIV-1 infection is initiated through a high-affinity interaction between the HIV-1 external envelope glycoprotein (gp120) and the CD4 receptor on T cells. Cell killing is a later event intimately linked by in vitro genetic analyses with the fusogenic properties of the HIV envelope glycoprotein gp120 and transmembrane glycoprotein gp41. In this report, we describe aberrancies in cell cycle regulatory proteins initiated by cell-cell contact between T cells expressing HIV-1 envelope glycoproteins and other T cells expressing CD4 receptors. Cells rapidly accumulate cyclin B protein and tyrosine-hyperphosphorylated p34cdc2 (cdk1) kinase, indicative of cell cycle arrest at G2 phase. Moreover, these cells continue to synthesize cyclin B protein, enlarge and display an abnormal ballooned morphology, and disappear from the cultures in a pattern previously described for cytotoxicity induced by DNA synthesis (S phase) inhibitors. Similar changes are observed in peripheral blood mononuclear cells infected in vitro with pathogenic primary isolates of HIV-1.

Coexpression of NF-kappa B/Rel and Sp1 transcription factors in human immunodeficiency virus 1-induced, dendritic cell-T-cell syncytia.

Productive infection of T cells with human immunodeficiency virus 1 (HIV-1) typically requires that the T cells be stimulated with antigens or mitogens. This requirement has been attributed to the activation of the transcription factor NF-kappa B, which synergizes with the constitutive transcription factor Sp1 to drive the HIV-1 promoter. Recently, we have found that vigorous replication of HIV-1 takes place in nonactivated memory T cells after syncytium formation with dendritic cells (DCs). These syncytia lack activated cells as determined by an absence of staining for Ki-67 cell cycle antigen. The expression and activity of NF-kappa B and Sp1 were, therefore, analyzed in isolated T cells and DCs from humans and mice. We have used immunolabeling, Western blot analysis, and electrophoretic mobility shift and supershift assays. T cells lack active NF-kappa B but express Sp1 as expected. DCs express high levels of all known NF-kappa B and Rel proteins, with activity residing primarily within RelB, p50, and p65. However, DCs lack Sp1, which may explain the failure of HIV-1 to replicate in purified DCs. Coexpression of NF-kappa B and Sp1 occurs in the heterologous DC-T-cell syncytia that are induced by HIV-1. Therefore, HIV-1-induced cell fusion brings together factors that upregulate virus transcription. Since DCs and memory T cells frequently traffic together in situ, these unusual heterologous syncytia could develop in infected individuals and lead to chronic HIV-1 replication without ostensible immune stimulation.

Hepatitis B virus HBx protein deregulates cell cycle checkpoint controls.

The human hepatitis B virus (HBV) HBx protein is a small transcriptional activator that is essential for virus infection. HBx is thought to be involved in viral hepatocarcinogenesis because it promotes tumorigenesis in transgenic mice. HBx activates the RAS-RAF-mitogen-activated protein (MAP) kinase signaling cascade, through which it activates transcription factors AP-1 and NF-kappa B, and stimulates cell DNA synthesis. We show that HBx stimulates cell cycle progression, shortening the emergence of cells from quiescence (G0) and entry into S phase by at least 12 h, and accelerating transit through checkpoint controls at G0/G1 and G2/M. Compared with serum stimulation, HBx was found to strongly increase the rate and level of activation of the cyclin-dependent kinases CDK2 and CDC2, and their respective active association with cyclins E and A or cyclin B. HBx is also shown to override or greatly reduce serum dependence for cell cycle activation. Both HBx and serum were found to require activation of RAS to stimulate cell cycling, but only HBx could shorten checkpoint intervals. HBx therefore stimulates cell proliferation by activating RAS and a second unknown effector, which may be related to its reported ability to induce prolonged activation of JUN or to interact with cellular p53 protein. These data suggest a molecular mechanism by which HBx likely contributes to viral carcinogenesis. By deregulating checkpoint controls, HBx could participate in the selection of cells that are genetically unstable, some of which would accumulate unrepaired transforming mutations.

Lack of functional retinoblastoma protein mediates increased resistance to antimetabolites in human sarcoma cell lines.

Growth inhibition assays indicated that the IC50 values for methotrexate (MTX) and 5-fluorodeoxyuridine (FdUrd) in HS-18, a liposarcoma cell line lacking retinoblastoma protein (pRB), and SaOS-2, an osteosarcoma cell line with a truncated and nonfunctional pRB, were 10- to 12-fold and 4- to 11-fold higher, respectively, than for the HT-1080 (fibrosarcoma) cell line, which has wild-type pRB. These Rb-/- cell lines exhibited a 2- to 4-fold increase in both dihydrofolate reductase (DHFR) and thymidylate synthase (TS) enzyme activities as well as a 3- to 4-fold increase in mRNA levels for these enzymes compared to the HT-1080 (Rb+/+) cells. This increase in expression was not due to amplification of the DHFR and TS genes. Growth inhibition by MTX and FdUrd was increased and DHFR and TS activities and expression were correspondingly decreased in Rb transfectants of SaOS-2 cells. In contrast, there was no significant difference in growth inhibition among these cell lines for the nonantimetabolites VP-16, cisplatin, and doxorubicin. A gel mobility-shift assay showed that parental SaOS-2 cells had increased levels of free E2F compared to the Rb-reconstituted SaOS-2 cells. These results indicate that pRB defective cells may have decreased sensitivity to growth inhibition by target enzymes encoded by genes whose transcription is enhanced by E2F proteins and suggest mechanisms of interaction between cytotoxic agents and genes involved in cell cycle progression.

Inhibition of the integrase of human immunodeficiency virus (HIV) type 1 by anti-HIV plant proteins MAP30 and GAP31.

MAP30 (Momordica anti-HIV protein of 30 kDa) and GAP31 (Gelonium anti-HIV protein of 31 kDa) are anti-HIV plant proteins that we have identified, purified, and cloned from the medicinal plants Momordica charantia and Gelonium multiflorum. These antiviral agents are capable of inhibiting infection of HIV type 1 (HIV-1) in T lymphocytes and monocytes as well as replication of the virus in already-infected cells. They are not toxic to normal uninfected cells because they are unable to enter healthy cells. MAP30 and GAP31 also possess an N-glycosidase activity on 28S ribosomal RNA and a topological activity on plasmid and viral DNAs including HIV-1 long terminal repeats (LTRs). LTRs are essential sites for integration of viral DNA into the host genome by viral integrase. We therefore investigated the effect of MAP30 and GAP31 on HIV-1 integrase. We report that both of these antiviral agents exhibit dose-dependent inhibition of HIV-1 integrase. Inhibition was observed in all of the three specific reactions catalyzed by the integrase, namely, 3' processing (specific cleavage of the dinucleotide GT from the viral substrate), strand transfer (integration), and "disintegration" (the reversal of strand transfer). Inhibition was studied by using oligonucleotide substrates with sequences corresponding to the U3 and U5 regions of HIV LTR. In the presence of 20 ng of viral substrate, 50 ng of target substrate, and 4 microM integrase, total inhibition was achieved at equimolar concentrations of the integrase and the antiviral proteins, with EC50 values of about 1 microM. Integration of viral DNA into the host chromosome is a vital step in the replicative cycle of retroviruses, including the AIDS virus. The inhibition of HIV-1 integrase by MAP30 and GAP31 suggests that impediment of viral DNA integration may play a key role in the anti-HIV activity of these plant proteins.

Identification of human cyclin-dependent kinase 8, a putative protein kinase partner for cyclin C.

Metazoan cyclin C was originally isolated by virtue of its ability to rescue Saccharomyces cerevisiae cells deficient in G1 cyclin function. This suggested that cyclin C might play a role in cell cycle control, but progress toward understanding the function of this cyclin has been hampered by the lack of information on a potential kinase partner. Here we report the identification of a human protein kinase, K35 [cyclin-dependent kinase 8 (CDK8)], that is likely to be a physiological partner of cyclin C. A specific interaction between K35 and cyclin C could be demonstrated after translation of CDKs and cyclins in vitro. Furthermore, cyclin C could be detected in K35 immunoprecipitates prepared from HeLa cells, indicating that the two proteins form a complex also in vivo. The K35-cyclin C complex is structurally related to SRB10-SRB11, a CDK-cyclin pair recently shown to be part of the RNA polymerase II holoenzyme of S. cerevisiae. Hence, we propose that human K35(CDK8)-cyclin C might be functionally associated with the mammalian transcription apparatus, perhaps involved in relaying growth-regulatory signals.

Transforming growth factor beta mediates the progesterone suppression of an epithelial metalloproteinase by adjacent stroma in the human endometrium.

Unlike most normal adult tissues, cyclic growth and tissue remodeling occur within the uterine endometrium throughout the reproductive years. The matrix metalloproteinases (MMPs), a family of structurally related enzymes that degrade specific components of the extracellular matrix are thought to be the physiologically relevant mediators of extracellular matrix composition and turnover. Our laboratory has identified MMPs of the stromelysin family in the cycling human endometrium, implicating these enzymes in mediating the extensive remodeling that occurs in this tissue. While the stromelysins are expressed in vivo during proliferation-associated remodeling and menstruation-associated endometrial breakdown, none of the stromelysins are expressed during the progesterone-dominated secretory phase of the cycle. Our in vitro studies of isolated cell types have confirmed progesterone suppression of stromal MMPs, but a stromal-derived paracrine factor was found necessary for suppression of the epithelial-specific MMP matrilysin. In this report, we demonstrate that transforming growth factor beta (TGF-beta) is produced by endometrial stroma in response to progesterone and can suppress expression of epithelial matrilysin independent of progesterone. Additionally, we find that an antibody directed against the mammalian isoforms of TGF-beta abolishes progesterone suppression of matrilysin in stromal-epithelial cocultures, implicating TGF-beta as the principal mediator of matrilysin suppression in the human endometrium.

De novo decorin gene expression suppresses the malignant phenotype in human colon cancer cells.

The rapid progress in the cloning of proteoglycan genes has enabled investigators to examine in depth the functional roles these polyhedric molecules play in the control of cell proliferation. Decorin, a leucine-rich proteoglycan expressed by most connective tissues, is a prototype molecule that regulates cellular growth via two mechanisms: modulation of growth factor activity and matrix assembly. We now provide direct evidence that human colon cancer cells stably transfected with decorin cDNA exhibit a marked suppression of the transformed phenotype: the cells have a reduced growth rate in vitro, form small colonies in soft agar, and do not generate tumors in scid/scid mice. Several independent clones are arrested in the G1 phase of the cell cycle, and their growth suppression can be restored by treatment with decorin antisense oligodeoxynucleotides. These effects are independent of growth factors and are not due to either clonal selection or integration site of the decorin gene. These findings correlate well with the observation that decorin gene expression is markedly up-regulated during quiescence. Decorin thus appears to be one component of a negative loop that controls cell growth.

Exit from G0 and entry into the cell cycle of cells expressing p21Sdi1 antisense RNA.

p21Sdi1 (also known as Cip1 and Waf1), an inhibitor of DNA synthesis cloned from senescent human fibroblasts, is an inhibitor of G1 cyclin-dependent kinases (Cdks) in vitro and is transcriptionally regulated by wild-type p53. In addition, p21Sdi1 has been found to inhibit DNA replication by direct interaction with proliferating cell nuclear antigen. In this study we analyzed normal human fibroblast cells arrested in G0 and determined that an excess of p21Sdi1 was present after immunodepletion of various cyclins and Cdks, in contrast to mitogen-stimulated cells in early S phase. Expression of antisense p21Sdi1 RNA in G0-arrested cells resulted in induction of DNA synthesis as well as entry into mitosis. These results suggest that p21Sdi1 functions in G0 and early G1 and that decreased expression of the gene is necessary for cell cycle progression.

Phosphorylated and unphosphorylated forms of human single-stranded DNA-binding protein are equally active in simian virus 40 DNA replication and in nucleotide excision repair.

The trimeric human single-stranded DNA-binding protein (HSSB; also called RP-A) plays an essential role in DNA replication, nucleotide excision repair, and homologous DNA recombination. The p34 subunit of HSSB is phosphorylated at the G1/S boundary of the cell cycle or upon exposure of cells to DNA damage-inducing agents including ionizing and UV radiation. We have previously shown that the phosphorylation of p34 is catalyzed by both cyclin-dependent kinase-cyclin A complex and DNA-dependent protein kinase. In this study, we investigated the effect of phosphorylation of p34 by these kinases on the replication and repair function of HSSB. We observed no significant difference with the unphosphorylated and phosphorylated forms of HSSB in the simian virus 40 DNA replication or nucleotide excision repair systems reconstituted with purified proteins. The phosphorylation status of the p34 subunit of HSSB was unchanged during the reactions. We suggest that the phosphorylated HSSB has no direct effect on the basic mechanism of DNA replication and nucleotide excision repair reactions in vitro, although we cannot exclude a role of p34 phosphorylation in modulating HSSB function in vivo through a yet poorly understood control pathway in the cellular response to DNA damage and replication.

The retinoblastoma-susceptibility gene product binds directly to the human TATA-binding protein-associated factor TAFII250.

RB, the protein product of the retinoblastoma tumor-suppressor gene, regulates the activity of specific transcription factors. This regulation appears to be mediated either directly through interactions with specific transcription factors or through an alternative mechanism. Here we report that stimulation of Sp1-mediated transcription by RB is partially abrogated at the nonpermissive temperature in ts13 cells. These cells contain a temperature-sensitive mutation in the TATA-binding protein-associated factor TAFII250, first identified as the cell cycle regulatory protein CCG1. The stimulation of Sp1-mediated transcription by RB in ts13 cells at the nonpermissive temperature could be restored by the introduction of wild-type human TAFII250. Furthermore, we demonstrate that RB binds directly to hTAFII250 in vitro and in vivo. These results suggest that RB can confer transcriptional regulation and possibly cell cycle control and tumor suppression through an interaction with TFIID, in particular with TAFII250.