Antiproliferative Signals Research Articles

A 1-Mb Physical Map and PAC Contig of the Imprinted Domain in 11p15.5 That Contains TAPA1 and the BWSCR1/WT2 Region

We have constructed a 1-Mb contig in human chromosomal band 11p15.5, a region implicated in the etiology of several embryonal tumors, including Wilms tumor, and in Beckwith–Wiedemann syndrome. Cosmid, P1, PAC, and BAC clones were characterized byNotI/SalI digestion and hybridized to a variety of probes to generate a detailed physical map that extends from D11S517 to L23MRP. Included in the map are the CARS, NAP2, p57/KIP2, KVLQT1, ASCL2, TH, INS, IGF2, H19, and L23MRP genes as well as end probes isolated from PACs. The TAPA1 gene, whose protein product can transmit an antiproliferative signal, was also localized in the contig. However, Northern blot analysis demonstrated that its expression did not correlate with tumorigenicity in G401 Wilms tumor hybrids, suggesting that TAPA1 is not responsible for the tumor suppression associated with 11p15.5. Genomic clones were used as probes in FISH analysis to map the breakpoints from three Beckwith–Wiedemann syndrome patients and a rhabdoid tumor. Interestingly, each of the breakpoints disrupts the KVLQT1 gene, which is spread over a 400-kb region of the contig. Since 11p15.5 contains several genes with imprinted expression and one or more tumor suppressor genes, our contig and map provide a framework for characterizing this intriguing genetic environment.

Early Growth Response-1-dependent Apoptosis Is Mediated by p53

The early growth response-1 (EGR-1) protein is an anti-proliferative signal for certain tumor cells and is required for apoptosis induced by stimuli that elevate intracellular Ca2+. We present evidence that EGR-1 transactivates the promoter of the p53 gene and up-regulates p53 RNA and protein levels. Inhibition of p53 function with dominant-negative p53 mutants abrogates EGR-1-dependent apoptosis. These findings establish a direct functional link between EGR-1 and the p53-mediated cell death pathway and suggest that mutant forms of p53 in tumor cells may provide resistance to the anti-proliferative effects of EGR-1.

Coupling of epidermal growth factor (EGF) with the antiproliferative activity of cAMP induces neuronal differentiation.

Nerve growth factor (NGF) functions as a progression factor with both mitogenic and antimitogenic activities. When PC12 cells are treated with NGF, they advance to the G1 stage of the cell cycle before they differentiate. The correlation between cessation of proliferation and differentiation suggests that the antimitotic activity of NGF may be obligatory for differentiation. Although epidermal growth factor- (EGF) and NGF-treated PC12 cells share several common properties, including activation of the mitogen-activated protein (MAP) kinase pathway and induction of immediate early genes, EGF is mitogenic for PC12 cells and does not normally stimulate differentiation. However, combinations of EGF and low levels of cAMP stimulate differentiation even though neither agent alone does (Mark, M. D., Liu, Y., Wong, S. T., Hinds, T. R., and Storm, D.R. (1995) J. Cell Biol. 130, 701-710). Since EGF is mitogenic for PC12 cells and differentiation may not occur until proliferation is inhibited, differentiation caused by cAMP and EGF may be due to the antiproliferative activity of cAMP. To test this hypothesis, we examined the effect of EGF or combinations of EGF and cAMP on PC12 cell proliferation. EGF alone stimulated proliferation of PC12 cells and increased the levels of several cell cycle progression factors including cdk2, cdk4, and cyclin B1. Cyclic AMP inhibited the EGF-stimulated increases in cell cycle progression factors as well as proliferation. Other antiproliferative agents including rapamycin, mimosine, and nitric oxide agonists also synergized with EGF to stimulate differentiation. These data indicate that the coupling of antiproliferative signals with EGF modifies the biological properties of EGF and converts it to a differentiating growth factor.

A proline-rich motif in p53 is required for transactivation-independent growth arrest as induced by Gas1.

The involvement of p53 in regulating diverse cellular processes dictates that it must respond to multiple signaling mechanisms, thus coordinating the response to various "stress conditions." Genotoxic stress has served as a paradigm to dissect the transactivation-dependent branch of the pathway by which p53 can induce growth arrest. Alternate mechanisms have been invoked to explain transactivation-independent effects of p53, especially in the context of apoptosis. We have identified a p53-dependent pathway initiated by the gas1 product, a plasma membrane protein highly expressed during G0, which activates a transactivation-independent p53 growth arrest function. Through a detailed deletional analysis and site-specific mutagenesis of p53 we show that the Gas1-dependent signal transduction relies on a proline-rich region (amino acids 63-85) of murine p53. In vivo competition experiments using combinations of such mutants implicate this functional domain of p53 as a docking site in the transmission of antiproliferative signals.

CD30 Ligand Is Frequently Expressed in Human Hematopoietic Malignancies of Myeloid and Lymphoid Origin

AbstractCD30 ligand (CD30L) is a type-II membrane glycoprotein capable of transducing signals leading to either cell death or proliferation through its specific counterstructure CD30. Although several lines of evidence indicate that CD30L plays a key role as a paracrine- or autocrine-acting surface molecule in the deregulated cytokine cascade of Hodgkin's disease, little is known regarding its distribution and biologic significance in other human hematopoietic malignancies. By analyzing tumor cells from 181 patients with RNA studies and immunostaining by the anti-CD30L monoclonal antibody M80, we were able to show that human hematopoietic malignancies of different lineage and maturation stage display a frequent and broad expression of the ligand. CD30L mRNA and surface protein were detected in 60% of acute myeloid leukemias (AMLs), 54% of B-lineage acute lymphoblastic leukemias (ALLs), and in a consistent fraction (68%) of B-cell lymphoproliferative disorders. In this latter group, hairy cell leukemia and high-grade B-cell non-Hodgkin's lymphoma (B-NHL) expressed a higher surface density of CD30L as compared with B-cell chronic lymphocytic leukemia and low-grade B-NHL. Purified plasmacells from a fraction of multiple myeloma patients also displayed CD30L mRNA and protein. A more restricted expression of CD30L was found in T-cell tumors that was mainly confined to neoplasms with an activated peripheral T-cell phenotype, such as T-cell prolymphocytic leukemia, peripheral T-NHL, and adult T-cell leukemia/lymphoma. In contrast, none of the T-lineage ALLs analyzed expressed the ligand. In AML, a high cellular density of CD30L was detected in French-American-British M3, M4, and M5 phenotypes, which are directly associated with the presence on tumor cells of certain surface structures, including the p55 interleukin-2 receptor α-chain, the αM (CD11b) chain of β2 integrins, and the intercellular adhesion molecule-1 (CD54). Analysis of normal hematopoietic cells evidenced that, in addition to circulating and tonsil B cells, a fraction of bone marrow myeloid precursors, erythroblasts, and subsets of megakaryocytes also express CD30L. Finally, we have shown that native CD30L expressed on primary leukemic cells is functionally active by triggering both mitogenic and antiproliferative signals on CD30+ target cells. As opposed to CD30L, only 10 of 181 primary tumors expressed CD30 mRNA or protein, rendering therefore unlikely a CD30-CD30L autocrine loop in human hematopoietic neoplasms. Taken together, our data indicate that CD30L is widely expressed from early to late stages of human hematopoiesis and suggest a regulatory role for this molecule in the interactions of normal and malignant hematopoietic cells with CD30+ immune effectors and/or microenvironmental accessory cells.

Mitogenic and anti-proliferative signals for neural crest cells and the neurogenic action of TGF-beta1.

The influence of pertinent growth factors on proliferation and differentiation of quail neural crest cell was assessed by in vitro colony assay in a serum-free (0.5% chick embryo-extract supplemented) culture medium. The factors tested included basic fibroblast growth factor (bFGF; FGF-2), neurotrophins, and transforming growth factor-beta-1 (TGF-beta). Both bFGF and neurotrophins are implicated in the development of the peripheral nervous system, whereas TGF-beta can affect cell differentiation and modulate the action of other growth factors. Bromodeoxyuridine (BrdU) incorporation indicated that bFGF is mitogenic to pluripotent neural crest cells (and/or their immediate progeny) and to committed melanogenic cells. However, this was not reflected in an increase in colony size. In contrast, colony size did increase when nerve growth factor (NGF) was present in addition to bFGF. This indicated either that both factors are required to initiate cell proliferation or that at least some bFGF-exposed cells become dependent on neurotrophins for survival. Sequential addition of the factors showed that exposure to bFGF was required prior to the presence of a neurotrophin, thus favoring the latter possibility. All three neurotrophins tested, NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), were capable of supporting survival of pluripotent neural crest cells (or their closely related progeny) in the presence of bFGF. In the absence of bFGF, neurotrophins did not affect colony size. Although the BrdU data indicated that bFGF is also a mitogen for committed melanogenic cells, the size of pigmented colonies did not change in the presence of bFGF alone or of bFGF plus a neurotrophin. This suggested that another, yet to be determined, factor is required for the survival of proliferating melanogenic cells. Colony assays were also performed in the presence and absence of TGF-beta, both alone and in combination with bFGF plus NGF. TGF-beta inhibited proliferation of both pluripotent neural crest cells (and/or their immediate derivatives) and of committed melanogenic cells, causing a decrease in colony size. When TGF-beta was added to the culture medium together with the bFGF/NGF combination, this also caused a significant decrease in colony size, similar to the one observed with TGF-beta alone. TGF-beta blocked proliferation even when the cells were exposed 24 to 48 hr to the bFGF/NGF combination prior to addition of TGF-beta. Neurogenesis increased significantly in the presence of TGF-beta. The number per colony of both adrenergic cells and sensory neuron precursors increased in TGF-beta-treated neuroblast-positive colonies. The following new insights were derived from this study: 1) basic FGF is a mitogen for pluripotent neural crest cells (and/or their immediate derivatives); 2) pluripotent and committed melanogenic neural crest cells that have been exposed to bFGF become dependent on trophic support; 3) all neurotrophins tested (NGF, BDNF or NT-3) can fulfill the trophic requirement of bFGF-exposed pluripotent cells, but not for melanogenic cells; 4) TGF-beta is an anti-proliferative signal for pluripotent neural crest cells and for committed melanogenic cells; 5) the TGF-beta-mediated anti-proliferative signal dominates over the bFGF/neurotrophin-mediated mitogenic signal; and 6) TGF-beta enhances sensory and adrenergic neurogenesis, possibly by acting upon a common neurogenic precursor cell. Furthermore, our work confirms previous reports by other investigators, who showed that bFGF promotes and TGF-beta inhibits proliferation of pigment cells.

Mitogenic and anti‐proliferative signals for neural crest cells and the neurogenic action of TGF‐β1

The influence of pertinent growth factors on proliferation and differentiation of quail neural crest cell was assessed by in vitro colony assay in a serum-free (0.5% chick embryo-extract supplemented) culture medium. The factors tested included basic fibroblast growth factor (bFGF; FGF-2), neurotrophins, and transforming growth factor-beta-1 (TGF-β). Both bFGF and neurotrophins are implicated in the development of the peripheral nervous system, whereas TGF-β can affect cell differentiation and modulate the action of other growth factors. Bromodeoxyuridine (BrdU) incorporation indicated that bFGF is mitogenic to pluripotent neural crest cells (and/or their immediate progeny) and to committed melanogenic cells. However, this was not reflected in an increase in colony size. In contrast, colony size did increase when nerve growth factor (NGF) was present in addition to bFGF. This indicated either that both factors are required to initiate cell proliferation or that at least some bFGF-exposed cells become dependent on neurotrophins for survival. Sequential addition of the factors showed that exposure to bFGF was required prior to the presence of a neurotrophin, thus favoring the latter possibility. All three neurotrophins tested, NGF, brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3), were capable of supporting survival of pluripotent neural crest cells (or their closely related progeny) in the presence of bFGF. In the absence of bFGF, neurotrophins did not affect colony size. Although the BrdU data indicated that bFGF is also a mitogen for committed melanogenic cells, the size of pigmented colonies did not change in the presence of bFGF alone or of bFGF plus a neurotrophin. This suggested that another, yet to be determined, factor is required for the survival of proliferating melanogenic cells. Colony assays were also performed in the presence and absence of TGF-β, both alone and in combination with bFGF plus NGF. TGF-β inhibited proliferation of both pluripotent neural crest cells (and/or their immediate derivatives) and of committed melanogenic cells, causing a decrease in colony size. When TGF-β was added to the culture medium together with the bFGF/NGF combination, this also caused a significant decrease in colony size, similar to the one observed with TGF-β alone. TGF-β blocked proliferation even when the cells were exposed 24 to 48 hr to the bFGF/NGF combination prior to addition of TGF-β. Neurogenesis increased significantly in the presence of TGF-β. The number per colony of both adrenergic cells and sensory neuron precursors increased in TGF-β-treated neuroblast-positive colonies. The following new insights were derived from this study: 1) basic FGF is a mitogen for pluripotent neural crest cells (and/or their immediate derivatives); 2) pluripotent and committed melanogenic neural crest cells that have been exposed to bFGF become dependent on trophic support; 3) all neurotrophins tested (NGF, BDNF or NT-3) can fulfill the trophic requirement of bFGF-exposed pluripotent cells, but not for melanogenic cells; 4) TGF-β is an anti-proliferative signal for pluripotent neural crest cells and for committed melanogenic cells; 5) the TGF-β-mediated anti-proliferative signal dominates over the bFGF/neurotrophin-mediated mitogenic signal; and 6) TGF-β enhances sensory and adrenergic neurogenesis, possibly by acting upon a common neurogenic precursor cell. Furthermore, our work confirms previous reports by other investigators, who showed that bFGF promotes and TGF-β inhibits proliferation of pigment cells. Dev. Dyn. 208:375–386, 1997. © 1997 Wiley-Liss, Inc.

Activation of the lymphotoxin beta receptor by cross-linking induces chemokine production and growth arrest in A375 melanoma cells.

The lymphotoxin beta receptor (LT beta R) was originally described as a transcribed sequence encoded on human chromosome 12p, with homology to the TNF receptor family. Subsequently, a recombinant LT beta R was shown to bind LT alpha LT beta heteromeric complexes. In this study, we have shown that LT beta R is expressed in a variety of tissues and cell lines of monocytic lineage, as well as in fibroblast and human melanoma cell lines. Unlike other members of the TNF receptor family, LT beta R is not expressed by peripheral blood T cells. A chimeric fusion protein consisting of the extracellular domain of LT beta R fused to the Fc region of human IgG1 was used to develop mAbs against LT beta R. Cross-linking LT beta R on A375 melanoma cells with these Abs generated an antiproliferative signal. In addition, the IL-8 and RANTES chemokines, early indicators of inflammation, were secreted by the A375 melanoma line and the WI38VA13 fibroblast line in response to cross-linking of LT beta R. These same activities could be induced by membrane-bound and soluble LT beta and LT alpha LT beta oligomers.

Octapeptide somatostatin analog SMS 201-995 induces translocation of intracellular PTP1C to membranes in MCF-7 human breast adenocarcinoma cells.

Somatostatin (SST) analogs exert direct antiproliferative actions in pancreatic, pituitary, and mammary tumor cells in vitro. SST receptor (SSTR)-mediated induction of membrane-associated protein tyrosine phosphatase (PTP) activity has been implicated in its anti-proliferative signaling by virtue of its ability to dephosphorylate and inactivate growth factor receptor kinases. Recently, a PTP-containing Src homology 2 domain, identified as PTP1C/SHPTP1/SHP/HCP, was found to be associated with SSTR in rat pancreatic acinar cell membranes. In the present study we investigated the antiproliferative action of the octapeptide SST analog SMS 201-995 (OCT) and its effect on PTP activity in MCF-7 human breast adenocarcinoma cells. We report here that OCT does not directly stimulate membrane-associated PTP activity, but induces translocation of intracellular PTP to the membrane in MCF-7 cells preincubated with the peptide in a time- and concentration-dependent manner. We demonstrate that this is due at least in part to OCT-induced recruitment of cytosolic PTP1C. OCT-induced recruitment of PTP1C to the cell surface as well as its ability to inhibit the growth of MCF-7 cells was G protein dependent and inhibited by orthovanadate. These findings suggest that translocation of cytosolic PTP1C by SST analogs to the cell surface is an early event in its antiproliferative signaling in tumor cells.

Viral oncogenesis and cell cycle control

Increasing evidence suggests a critical role for cell cycle regulatory genes in tumorigenesis in mammals, and oncogenic viruses account for a considerable proportion of cancers in humans. In most cases, infection by oncogenic viruses is insufficient for malignant transformation; rather, the viruses provide host cells with additional growth stimuli, which extend the proliferative capacity of the infected cell. This implies that viral oncogenes can override cellular control mechanisms, which in untransformed cells regulate cell cycle progression in response to various antiproliferative signals. Recent progress has enabled the definition of several mammalian cell cycle regulatory genes as targets for viral oncoproteins. This new field of investigation was the central topic of a meeting (Viral Oncogenesis and Cell Cycle Control) held in Heidelberg in October 1995. In the following, major findings presented at this meeting will be summarized. The first part of the meeting was dedicated to the analysis of cell cycle regulation in normal untransformed mammalian cells, followed by an analysis of the interaction of tumor virus oncoproteins with mammalian cell cycle regulators. The latter interactions fall into three categories: viral oncoproteins were shown to interfere with various signal transduction pathways, to inactivate growth-suppressive nuclear proteins by direct binding, and to modulate the expression of key cell cycle regulatory genes.

The WAF 1-mediated p53 growth-suppressor pathway is intact in the coronary arteries of heart transplant recipients

It has been suggested that the interaction of cytomegalovirus (CMV) with the p53 tumor suppressor gene product plays a role in the development of coronary artery restenosis after angioplasty. CMV nucleic acids have been observed in the coronary arteries of allografted hearts, suggesting a possible role for the interaction of CMV with p53 in the development of accelerated graft arteriosclerosis in transplant recipients. Formalin-fixed, paraffin-embedded sections of coronary arteries from 19 transplanted hearts were immunostained for the p53 gene product using Target Unmasking Fluid (TUF) - mediated immunohistochemistry and the anti-p53 antibodies CM1 and D07. Fresh-frozen sections of coronary arteries were also available from six of the 19 hearts, and these fresh-frozen sections were immunostained for the p53 gene product with the D07 antibody and for WAF1 using the anti-WAF1 antibody EA10. Focal and weak staining for p53 was observed in smooth muscle and endothelial cells in two of 19 vessels, whereas the remaining 17 did not stain. CMV nucleic acids were previously shown in six of 13 of these hearts by in situ hybridization. The fresh-frozen sections of coronary arteries also did not stain for p53, but the smooth muscle cells in these vessels did stain intensely for WAF1. These results suggest three possibilities: (1) CMV-p53 interactions are not important in the development of accelerated graft arteriosclerosis; or (2) there is an interaction, but it is transient and not detectable at the time points examined in this study; or (3) there is an interaction, but binding of CMV to p53 leads to accelerated degradation of p53, as occurs with HPV-E6. The expression of WAF1 further suggests that the WAF1-mediated antiproliferative signal is intact in these vessels.

Expression Cloning of an Interferon-inducible 17-kDa Membrane Protein Implicated in the Control of Cell Growth

Interferon-inducible membrane proteins of approximately 17 kDa have been suggested to play a role in the antiproliferative activity of interferons based on (1) their pattern of induction in interferon-sensitive and -resistant cell lines and (2) the ability of a membrane fraction enriched in 17-kDa proteins to inhibit cell growth. To gain insight into the nature of the proteins that mediate the antiproliferative activity of interferons, a monoclonal antibody, 13A5, was generated that reacted specifically with a 17-kDa interferon-inducible cell surface protein. The expression pattern of this 17-kDa protein by human cell lines correlated with sensitivity to the antiproliferative activity of interferons. To obtain information regarding the structure of this protein, the 13A5 antibody was used to screen COS cells transfected with a human cDNA expression library. Sequence analysis of a full-length cDNA clone revealed identity with the 9-27 cDNA, previously isolated on the basis of its interferon inducibility by differential screening. In addition, the 17-kDa protein encoded by the 9-27 gene was shown to be identical to the Leu-13 antigen. Leu-13 was previously identified as a 16-kDa interferon-inducible protein in leukocytes and endothelial cells and is a component of a multimeric complex involved in the transduction of antiproliferative and homotypic adhesion signals. These results suggest a novel level of cellular regulation by interferons involving a membrane protein, encoded by the interferon-inducible 9-27 gene, which associates with other proteins at the cell surface, forming a complex relaying growth inhibitory and aggregation signals.

Time, telomeres and tumours: is cellular senescence more than an anticancer mechanism?

Normal diploid cells, by definition, have a limited life span: they senesce after a set number of divisions both in vivo and in culture. It has been hypothesized that the molecular mechanism that measures the life span of a cell probably involves the shortening of telomeres that occurs with each round of DNA replication. This loss of telomeres is thought to induce antiproliferative signals that result in the induction of cellular senescence. In this article, Woodring Wright and Jerry Shay present a hypothesis for the mechanisms by which telomere shortening regulates cellular physiology and argue that cellular senescence is not only an anticancer mechanism but is also the cause of many of the degenerative changes of aging.

Transforming growth factor beta induces the cyclin-dependent kinase inhibitor p21 through a p53-independent mechanism.

The transforming growth factor beta s (TGF-beta s) are a group of multifunctional growth factors which inhibit cell cycle progression in many cell types. The TGF-beta-induced cell cycle arrest has been partially attributed to the regulatory effects of TGF-beta on both the levels and the activities of the G1 cyclins and their kinase partners. The activities of these kinases are negatively regulated by a number of small proteins, p21 (WAF1, Cip1), p27Kip1, p16, and p15INK4B, that physically associate with cyclins, cyclin-dependent kinases, or cyclin-Cdk complexes. p21 has been previously shown to be transcriptionally induced by DNA damage through p53 as a mediator. We demonstrate that TGF-beta also causes a rapid transcriptional induction of p21, suggesting that p21 can respond to both intracellular and extracellular signals for cell cycle arrest. In contrast to DNA damage, however, induction of p21 by TGF-beta is not dependent on wild-type p53. The cell line studied in these experiments, HaCaT, contains two mutant alleles of p53, which are unable to activate transcription from the p21 promoter when overexpressed. In addition, TGF-beta and p53 act through distinct elements in the p21 promoter. Taken together, these findings suggest that TGF-beta can induce p21 through a p53-independent pathway. Previous findings have implicated p27Kip1 and p15INK2B as effectors mediating the TGF-beta growth inhibitory effect. These results demonstrate that a single extracellular antiproliferative signal, TGF-beta, can act through multiple signaling pathways to elicit a growth arrest response.

Mammalian anti proliferative signals and their targets

The effect of mitogens on the mammalian cell cycle is opposed by the action of antimitogens, such as TGF-β, CAMP agonists, and various anti proliferative drugs. The recent identification of TGF-β receptors that initiate antimitogenic signals and of cell cycle kinase inhibitors that respond to these signals has provided new insights into this process. The evidence argues that mitogenic and antimitogenic signals confront each other by regulating in opposite ways the activity of cyclin-dependent kinases that control cell commitment to DNA replication.

Human T cells express specific binding sites for C1q. Role in T cell activation and proliferation.

Although the receptor that binds to the collagen-like domain of human C1q (C1qR) is expressed on a wide variety of cell types, the presence or absence of this receptor on human T lymphocytes has been debatable. The current studies were undertaken to re-examine whether human T cells possess specific binding sites for C1q by using a combination of techniques, including radioligand binding studies, flow cytometric analysis, and epifluorescence imaging techniques. Radioligand binding studies indicate that both peripheral T cells and the cultured T cell line, MOLT4, bind 125I-labeled C1q in a specific and apparently saturable manner, reaching equilibrium within 30 min at 37 degrees C under conditions of subphysiologic (90 mM NaCl) ionic strength. Western blot analysis with anti-C1qR of membrane proteins derived from Raji and MOLT4 cells showed an apparent single band of approximately 60 kDa under nonreducing conditions. Furthermore, when peripheral blood T cells were stimulated with 12,-o-tetradecanoyl phorbol-13-ester acetate for 5 days at 37 degrees C and assessed by FACS for their ability to bind anti-C1qR, the mitogen-induced cells were found to bind 40 to 50% more than their unstimulated counterparts. In addition, both CD4+ and CD8+ T cells were found to bind anti-C1qR. When the cells were mitogen induced with either 12,-o-tetradecanoyl phorbol-13-ester acetate, Con A, or PWM for 48 h in the presence or absence of 50 micrograms/ml C1q then pulsed with 1 microCi [3H]thymidine for 16 h at 37 degrees C, proliferation was significantly inhibited (40 to 80%, n = 7) as assessed by reduced [3H]thymidine incorporation. Taken together, the data suggest that: 1) Human T cells express C1qR in which immunoblots reveal a 60-kDa single chain protein. 2) C1qR expression is up-regulated by mitogens that induce T cell proliferation. 3) The primary ligand, C1q, induces an antiproliferative signal, which suggests that the C1qR plays a role in T cell activation and proliferation. In addition, the data contribute to the characterization of C1qRs on cells in peripheral blood and indicate that all cells, with the exception of erythrocytes, bear functional C1q receptors.

Close Correlation between the Dephosphorylation of p53 and Growth Suppression by Transforming Growth Factor‐β1 in Nasopharyngeal Carcinoma Cells Transduced with Adenovirus Early Region Genes

The mechanism of growth inhibition by transforming growth factor (TGF)‐β1 was investigated. We examined the growth inhibitory effects of TGF‐β1 on human nasopharyngeal carcinoma (KB) cells which constitutively expressed p53. TGF‐β1 suppressed the DNA synthesis of KB cells in a dose‐dependent manner. It had minimal effect on adenovirus‐2‐transduced KB cells expressing either adenovirus early region 1B (E1B) or 1A (E1A) product, which respectively binds to p53 or Rb product and inhibits its function, and no growth inhibition at all was observed with KB cells expressing both E1B and E1A products. Dephosphorylation of the p53 was promoted by TGF‐β1 stimulation in KB cells, but not in E1B‐producing KB cells, which sequestrate the function of p53. The growth inhibition of KB cells by TGF‐β1 was significantly reduced by treatment with okadaic acid. These results suggest that p53 transduces the antiproliferative signal of TGF‐β1 possibly through its dephosphorylation.

Anti-TAPA-1 antibodies induce protein tyrosine phosphorylation that is prevented by increasing intracellular thiol levels.

We studied the signal induced by the anti-TAPA-1 antibody and compared it to the signal induced by anti-IgM antibodies in a human B cell line, OCl-LY8. We found that exposure of these cells to either antibody resulted in a rapid increase in protein tyrosine phosphorylation which was prevented by inhibitors of tyrosine kinases. Tyrosine phosphorylation was an early event in the cascase leading to the antiproliferative effect of the anti-TAPA-1 antibody. However, 2-ME, a reducing agent that is not an inhibitor of tyrosine kinases, prevented both tyrosine phosphorylation and the antiproliferative effect of the antibody. Cells grown in low concentrations of 2-ME did not exhibit an increase in tyrosine phosphorylation in response to the anti-TAPA-1 antibody and were insensitive to the antiproliferative effect of the antibody. In contrast, the same cells maintained in 2-ME were able to induce tyrosine phosphorylation in response to anti-IgM. The use of 2-ME resulted in an increase in intracellular thiols, mostly glutathione. Moreover, compounds that block glutathione synthesis rendered cells susceptible to the antibody, even in the presence of 2-ME. These experiments demonstrate that tyrosine kinases are involved in propagating the antiproliferative signal initiated by the anti-TAPA-1 antibody and suggest that this signal is dependent upon the level of intracellular thiols.

Role of transforming growth factor-beta 1 in the cellular growth response to angiotensin II.

We have shown that angiotensin II (Ang II)-induced hypertrophy of vascular smooth muscle cells is dependent on the balance between proliferative and antiproliferative growth factors, specifically basic fibroblast growth factor and transforming growth factor-beta 1 (TGF-beta 1), respectively. We now present evidence, based on two phenotypically distinct cell cultures, that the ability to secrete the biologically active form of TGF-beta 1 is central to the growth response to Ang II. Two separate cultures were examined, one in which Ang II induces hypertrophy and the other in which Ang II induces hyperplasia. Ang II induces the expression of basic fibroblast growth factor twofold to fivefold in both cultures. Furthermore, both cultures express TGF-beta 1. In the culture that responds with hypertrophy, Ang II induces the expression of the active form of TGF-beta 1 twofold to threefold. However, in the culture that responds with hyperplasia, no active TGF-beta 1 was detected either at baseline or after Ang II exposure. Interestingly, all the TGF-beta 1 present was in the inactive, latent form. In the culture that responded with hyperplasia, Ang II induced a fourfold to fivefold increase in DNA synthesis. This increase could be abolished by the addition of active TGF-beta 1. Thus in these two cultures the ability to activate TGF-beta 1 dictates the cellular response to Ang II. These results support our hypothesis that a balance of proliferative and antiproliferative autocrine signals mediates the growth control of vascular smooth muscle cells.

Evidence for nuclear phosphoinositidase C activity in the antiproliferative signals produced by interferon in Burkitt lymphoma cells

The influence of interferon alpha on nuclear phosphoinositidase C (PIC) in Daudi cells has been analysed. Results showed an early increase of PIC activity detectable within 90 min of interferon treatment concomitant with an increase of diacylglycerol (DAG) levels. Since the interferon-induced DAG production is not modified by the addition of propranolol, a compound known to inhibit production of DAG from phosphatidylcholine hydrolysis, it is suggested that the interferon antiproliferative signal is transduced into the nucleus via the inositol lipid pathway. A parallel analysis performed on intact cells showed a rapid inhibition of PIC activity accompanied by an increase of DAG level thus suggesting that interferon-generated signals at plasma-membrane level use pathways different from that of inositol lipids. A selected clone of Daudi cells resistant to interferon action provided a control for specificity of results.