HIV-1 penetrates coronary artery endothelial cells by transcytosis.

BACKGROUND: The pathogenesis of HIV-1-related cardiomyopathy is poorly understood, but HIV-1 has been detected in cardiomyocytes. Whether HIV-1 penetrates into the myocardium by infection of coronary artery endothelial cells (CAEC) or using transcellular or paracellular routes across CAEC has not been resolved. MATERIALS AND METHODS: A model of the CAEC barrier was constructed with primary CAEC (derived from human coronary vessels). Polymerase chain reaction (PCR) assay, infectious assay, and immunofluorescence were employed to show abortive nature of HIV-1 infection of CAEC. Tight junction (TJ) and cell adhesion proteins were visualized by immunofluorescence. The time course of HIV-1 invasion was measured by HIV-1 RNA assay. Inulin permeability assay determined paracellular leakage. Transmission electron microscopy demonstrated virus-induced endothelial vacuolization. RESULTS: Despite a strong display on CAEC of CXCR4 and a lesser expression of CCR3 and CCR5, HIV-1 did not productively replicate in CAEC, as shown by infectious assay, immunofluorescence, and electron microscopy. HIV-1 infection of CAEC was abortive with minimal reverse transcription of strong stop DNA and pol but not full-length or two LTR DNA circles. Upon infection of the model with 1 million RNA copies of HIV-1JR-FL, virus penetration 2 hr postinfection (PI) was negligible but increased by 1,750% 24 hr PI. The paracellular permeability increased during this period by only 25%. Neither AOP-RANTES nor v-MIPII significantly reduced HIV-1JR-FL invasion. Virus infection did not alter the integral TJ protein occludin and the TJ-associated protein ZO-1. HIV-1 exposed CAEC and brain microvascular endothelial cells (BMVEC) developed extensive cytoplasmic vacuolization with retroviral-like particles in the vacuoles. CONCLUSIONS: The endothelium is not an impenetrable barrier to HIV-1. The virus opens a transcellular route across coronary and brain endothelia in cytoplasmic vacuoles.

Inhibition of CD3/CD28-mediated activation of the MEK/ERK signaling pathway represses replication of X4 but not R5 human immunodeficiency virus type 1 in peripheral blood CD4(+) T lymphocytes.

Binding of human immunodeficiency virus type 1 (HIV-1) to CD4 receptors induces multiple cellular signaling pathways, including the MEK/ERK cascade. While the interaction of X4 HIV-1 with CXCR4 does not seem to activate this pathway, viruses using CCR5 for entry efficiently activate MEK/ERK kinases (W. Popik, J. E. Hesselgesser, and P. M. Pitha, J. Virol. 72:6406-6413, 1998; W. Popik and P. M. Pitha, Virology 252:210-217, 1998). Since the importance of MEK/ERK in the initial steps of viral replication is poorly understood, we have examined the role of MEK/ERK signaling in the CD3- and CD28 (CD3/CD28)-mediated activation of HIV-1 replication in resting peripheral blood CD4(+) T lymphocytes infected with X4 or R5 HIV-1. We have found that the MEK/ERK inhibitor U0126 selectively inhibited CD3/CD28-stimulated replication of X4 HIV-1, while it did not affect the replication of R5 HIV-1. Inhibition of the CD3/CD28-stimulated MEK/ERK pathway did not affect the formation of the early proviral transcripts in cells infected with either X4 or R5 HIV-1, indicating that virus reverse transcription is not affected in the absence of MEK/ERK signaling. In contrast, the levels of nuclear provirus in cells infected with X4 HIV-1, detected by the formation of circular proviral DNA, was significantly lower in cells stimulated in the presence of MEK/ERK inhibitor than in the absence of the inhibitor. However, in cells infected with R5 HIV-1, the inhibition of the MEK/ERK pathway did not affect nuclear localization of the proviral DNA. These data suggest that the nuclear import of X4, but not R5, HIV-1 is dependent on a CD3/CD28-stimulated MEK/ERK pathway.

Binding of human immunodeficiency virus type 1 to CD4 and CXCR4 receptors differentially regulates expression of inflammatory genes and activates the MEK/ERK signaling pathway.

We have previously shown that binding of human immunodeficiency virus type 1 (HIV-1) virions to CD4 receptors stimulates association of Lck with Raf-1 and results in the activation of Raf-1 kinase in a Ras-independent manner. In the present study, we demonstrate that HIV-1 envelope glycoproteins of both T-cell-tropic and macrophagetropic strains rapidly activate the ERK/mitogen-activated protein (MAP) kinase pathway and the binding of nuclear transcription factors (AP-1, NF-kappaB, and C/EBP) and stimulate expression of cytokine and chemokine genes. The activation of this signaling pathway requires functional CD4 receptors and is independent of binding to CXCR4. Binding of the natural ligand stromal cell-derived factor 1 (SDF-1) to CXCR4, which inhibits entry of T-cell-tropic HIV-1, activates also the ERK/MAP kinase pathway. However, SDF-1 did not affect the CD4-mediated expression of cytokine and chemokine genes. These results provide firm molecular evidence that binding of HIV-1 envelope glycoproteins to CD4 receptor initiates a signaling pathway(s) independent of the binding to the chemokine receptor that leads to the aberrant expression of inflammatory genes and may contribute significantly to HIV-1 replication as well as to deregulation of the immune system.

Early activation of mitogen-activated protein kinase kinase, extracellular signal-regulated kinase, p38 mitogen-activated protein kinase, and c-Jun N-terminal kinase in response to binding of simian immunodeficiency virus to Jurkat T cells expressing CCR5 receptor.

We have shown that the binding of simian immunodeficiency virus (SIV) to Jurkat T cells expressing CD4 receptor strongly induces mitogen-activated protein (MAP) kinase kinase (MEK) and extracellular signal-regulated kinases 1 and 2 (ERK1/2) and only weakly induces p38 MAP kinase and c-Jun N-terminal kinase (JNK). Similarly, T-tropic NL4-3 virus, which uses both CD4 and CXCR4 receptors for entry, stimulated in these cells the MEK/ERK MAP kinase (MAPK) pathway in a CD4 receptor-dependent manner (Popik and Pitha, 1998). In contrast, both macrophage-tropic SIVmac316 and T cell-tropic SIVmac239, which in addition to CD4 require CCR5 coreceptor for entry, significantly enhanced early MEK/ERK, p38 MAPK, and JNK signaling in Jurkat cells expressing constitutively or transiently the CCR5 receptor. Together, this study provides the evidence that viruses using CXCR4 or CCR5 receptors for entry may differentially use signaling properties of their specific coreceptors to stimulate MAP kinase cascades. In addition, although SIVmac239 and SIVmac316 use different structural domains of the CCR5 receptor for entry, both viruses stimulate early phosphorylation of MEK, ERK, p38, and JNK independently of their tropism and replication.

Binding of human immunodeficiency virus type 1 to CD4 induces association of Lck and Raf-1 and activates Raf-1 by a Ras-independent pathway.

We have analyzed CD4-mediated signaling during the early stages of human immunodeficiency virus type 1 (HIV-1) infection. Binding of purified HIV-1 virions or recombinant HIV-1 glycoprotein gp120 to CD4 receptors resulted in association and tyrosine phosphorylation and activation of tyrosine kinase Lck and serine/threonine kinase Raf-1. The association between Lck and Raf-1 was mediated by stimulation of the CD4 receptors, since it was abolished by preincubation of the virus with soluble CD4 and was not detected in CD4-negative A201 T cells. However, the Lck-Raf-1 association was restored in A201 cells permanently transfected with human CD4 cDNA and stimulated with anti-CD4 antibodies. In addition, a catalytically active Lck was required for the association of Lck and Raf-1. Surprisingly, the CD4-mediated signaling, induced by the HIV-1 binding, did not result in stimulation of the Ras GTP-binding activity or its association with Raf-1, indicating that the signaling pathway generated by the HIV-1 binding is not identical to the classical Ras/Raf-1 pathway. Furthermore, overexpression of activated Raf-1 in Jurkat T cells stimulated the HIV long terminal repeat promoter activity and significantly enhanced HIV-1 replication. This suggests that the Lck-Raf-1 pathway, rapidly stimulated by the binding of HIV-1 or gp120 to CD4 receptors, may play an essential role in the transcriptional activation of the integrated HIV-1 provirus as well as in its pathogenicity.

Inhibition of human immunodeficiency virus type 1 replication by a Tat-activated, transduced interferon gene: targeted expression to human immunodeficiency virus type 1-infected cells.

We have examined the feasibility of using interferon (IFN) gene transfer as a novel approach to anti-human immunodeficiency virus type 1 (HIV-1) therapy in this study. To limit expression of a transduced HIV-1 long terminal repeat (LTR)-IFNA2 (the new approved nomenclature for IFN genes is used throughout this article) hybrid gene to the HIV-1-infected cells, HIV-1 LTR was modified. Deletion of the NF-kappa B elements of the HIV-1 LTR significantly inhibited Tat-mediated transactivation in T-cell lines, as well as in a monocyte line, U937. Replacement of the NF-kappa B elements in the HIV-1 LTR by a DNA fragment derived from the 5'-flanking region of IFN-stimulated gene 15 (ISG15), containing the IFN-stimulated response element, partially restored Tat-mediated activation of LTR in T cells as well as in monocytes. Insertion of this chimeric promoter (ISG15 LTR) upstream of the human IFNA2 gene directed high levels of IFN synthesis in Tat-expressing cells, while this promoter was not responsive to tumor necrosis factor alpha-mediated activation. ISG15-LTR-IFN hybrid gene inserted into the retrovirus vector was transduced into Jurkat and U937 cells. Selected transfected clones produced low levels of IFN A (IFNA) constitutively, and their abilities to express interleukin-2 and interleukin-2 receptor upon stimulation with phytohemagglutinin and phorbol myristate acetate were retained. Enhancement of IFNA synthesis observed upon HIV-1 infection resulted in significant inhibition of HIV-1 replication for a period of at least 30 days. Virus isolated from IFNA-producing cells was able to replicate in the U937 cells but did not replicate efficiently in U937 cells transduced with the IFNA gene. These results suggest that targeting IFN synthesis to HIV-1-infected cells is an attainable goal and that autocrine IFN synthesis results in a long-lasting and permanent suppression of HIV-1 replication.

Modulation of interferon-mediated inhibition of human immunodeficiency virus type 1 by Tat.

Recently, we have shown that in acutely infected T cells interferons (IFNs) effectively inhibit the human immunodeficiency type 1 (HIV-1) proviral DNA synthesis during a single replication cycle. In the present study, we have evaluated the relative effectiveness of IFNs in restricting HIV-1 expression at post-transcriptional level. Treatment of HeLa cells with IFNs A* and B (up to 1,000 U/ml) did not result in a reduction in HIV-1 RNA and protein synthesis encoded by the transfected HIV-1 proviral clone. Interestingly, IFN treatment reduced significantly the HIV-1 mRNA levels encoded by the transfected tat-defective HIV-1 provirus, and this inhibition could be overcome by transfection with Tat- and Rev-expressing plasmids. These results suggest that HIV-1-encoded Tat and Rev can overcome the inhibitory effects of IFNs on HIV-1 replication.

Differential effect of tumor necrosis factor-alpha and herpes simplex virus type 1 on the Tat-targeted inhibition of human immunodeficiency virus type 1 replication.

In this study, we have examined whether the Tat antagonist can inhibit human immunodeficiency virus type 1 (HIV-1) replication in the presence of cofactors that can activate transcription of HIV-1 provirus by an NF-kappa B-mediated mechanism, such as tumor necrosis factor-alpha (TNF-alpha) or herpes simplex virus type 1 (HSV-1) infection. As a prototype, we have chosen a low-molecular-weight Tat inhibitor, Ro5-3335, and analyzed its effect on HIV-1 replication in the presence of TNF-alpha and HSV-1 infection in acutely infected peripheral blood lymphocytes (PBLs) and T cells. Ro5-3335 inhibited HIV-1 replication both in CEM-174 cells and in PBLs, but the magnitude of the inhibition was inversely related to viral inoculum and the inhibition was only temporary; viral replication resumed at later times postinfection in spite of the continuous presence of the drug. In contrast, Ro5-3335 suppressed TNF-alpha-induced activation of HIV-1 replication in chronically infected T cells and monocytes that both expressed only low levels of HIV-1 constitutively, while its effect in high-expressing OM-10.1 cells was negligible in the presence of TNF-alpha. The inhibition of HIV-1 replication by Ro5-3335 was specific for the Tat-mediated effect and this drug was not able to inhibit the TNF-alpha-induced expression of the tat-defective HIV-1 provirus. In contrast to TNF-alpha, HSV-1-stimulated HIV-1 expression in the ACH-2 cells was effectively inhibited in the presence of Ro5-3335. These results demonstrate that Tat plays an essential role in HSV-1-mediated activation of HIV-1 provirus, while the TNF-alpha complementation of Tat shows cell-type specificity. These observations suggest that inhibition of the Tat function alone may not be sufficient for an effective anti-HIV-1 inhibition.

Evidence that levels of the dimeric cellular transcription factor CP2 play little role in the activation of the HIV-1 long terminal repeat in vivo or following superinfection with herpes simplex virus type 1.

The dimeric transcription factor CP2 binds a sequence element found near the transcription start site of the human immunodeficiency virus (HIV-1) long terminal repeat. Several groups have suggested that cellular factors binding this element might play a role in modulating HIV-1 promoter activity in vivo. For example, induction of latent HIV-1 gene expression in response to superinfection by herpes simplex virus type 1 (HSV-1) or cytomegalovirus is thought to be mediated, in part, by factors binding the CP2 site. In this report we began to examine directly the relationship between CP2 and expression of the HIV-1 promoter. First, we tested what effect HSV-1 infection of T cells had on the cellular levels of CP2. The results showed that HSV-1 infection led to a significant reduction in the level of CP2 DNA binding activity and protein within 20 h. Next, we tested the effect of overexpressing either the wild-type factor or a dominant negative variant of CP2 on HIV-1 promoter activity in vivo. The results showed that CP2 had little effect or slightly repressed HIV-1 promoter activity in vivo. In addition, these expression constructs had little effect on the induction of HIV-1 promoter activity elicited by HSV-1 infection.

The presence of tat protein or tumor necrosis factor alpha is critical for herpes simplex virus type 1-induced expression of human immunodeficiency virus type 1.

Tat-independent transcription of human immunodeficiency virus type 1 (HIV-1) plays an important role in virus life cycle before biologically significant levels of Tat protein have been accumulated. Using a latently infected T-cell line containing an integrated Tat-defective HIV-1 provirus, we examined whether factors known to up-regulate the HIV-1 expression in vitro can replace the requirement for a functional Tat protein and induce the expression of the Tat-defective HIV-1 provirus. Both tumor necrosis factor alpha (TNF-alpha) and herpes simplex virus type 1 (HSV-1) infection stimulated transcription of the Tat-defective HIV-1 provirus to comparable levels, but in HSV-1-infected cells, the cytoplasmic HIV-1 transcripts were not efficiently translated in the absence of Tat protein and were excluded from the large polysomes. However, HSV-1 infection did not affect the distribution of cellular gamma-actin RNA or 28S RNA in the polysomal fractions. The translational block of HIV-1 RNA was not mediated by the virion-associated host cell shutoff protein (vhs); dissociation of HIV-1 transcripts from the polysomes and inefficient translation was also observed in cells infected with the vhs-defective mutant of HSV-1 (vhs-1). Overexpression of Rev protein did not rescue the synthesis of HIV-1 proteins in these cells; however, the observed inhibition of HIV-1 RNA translation was efficiently overcome in the presence of Tat protein or TNF-alpha. These findings suggest that, in contrast to TNF-alpha, HSV-1 infection is not able to induce a full cycle of HIV-1 replication and that cytokines and Tat have a critical role in the activation of HIV-1 provirus by HSV-1.

Role of tumor necrosis factor alpha in activation and replication of the tat-defective human immunodeficiency virus type 1.

Transcription of human immunodeficiency virus type 1 (HIV-1) depends on the function of the virus-encoded regulatory protein Tat, which interacts with the specific Tat response (TAR) element present in the leader sequence of all HIV-1 RNAs. In this study, we examined whether tumor necrosis factor alpha (TNF-alpha) can replace the requirement for a functional Tat protein. We found that TNF-alpha can induce expression of a latent, tat-defective virus and support its replication both in T cells and in primary mononuclear cells. Analysis of the transcriptional rate of the tat-defective HIV-1 transcriptional unit indicates that TNF-alpha stimulates the initiation of transcription but, in contrast to Tat protein, does not significantly reduce transcriptional polarity. Interestingly, we found that the processing of viral precursor proteins is altered in the absence of Tat. We propose that TNF-alpha-mediated induction of HIV-1 plays an essential role in the early stages of the virus life cycle and in viral latency.

Transcriptional activation of the tat-defective human immunodeficiency virus type-1 provirus: effect of interferon.

The effect of human interferon-alpha 2 (HuIFN-alpha 2) on the activation of HIV-1 provirus was studied in cell lines containing either an integrated tat-defective HIV-1 provirus (HIV-1 (-tat)) (HNHIVdt4 cells) or the HIV-1 (-tat) provirus and a plasmid in which the expression of HuIFN-alpha 2 was under the control of HIV LTR (HNHIV alpha 1 cells). In both cell lines, the expression of HIV-1 RNA was below the limit of detection, but transcription of the HIV-1 (-tat) provirus could be induced either by transfection with Tat-expressing plasmid or by treatment with TPA and cycloheximide (CHX). By contrast, stimulation with TPA alone induced HIV-1 transcription only in HNHIVdt4 cells, but not in HNHIV alpha 1 cells that produced low levels of IFN-alpha constitutively. Similarly in a transient expression assay, TPA upregulated transcription of the transfected HIV-1 CAT plasmid only in HNHIVdt4 cells, but not in HNHIV alpha 1 cells. UV-crosslinking analysis of NF-kappa B-specific proteins induced in TPA-treated cells showed the presence of 45 and 55 kDa NF-kappa B-binding protein in TPA-induced HNHIVdt4 cells while, in HNHIV alpha 1 cells, we detected only 55-, 110-, and 200-kDa proteins, but no 45-kDa protein. The transcriptional effects of IFN could not, however, be seen in the presence of Tat protein, suggesting that the virus developed a mechanism to overcome the IFN-mediated restrictions.

Inhibition by interferon of herpes simplex virus type 1-activated transcription of tat-defective provirus.

The herpes simplex virus type 1 (HSV-1)-mediated transactivation of human immunodeficiency virus type 1 (HIV-1) provirus was studied in cell lines containing either integrated tat-defective HIV-1 provirus (HNHIVdt4 cells) or the tat-defective HIV-1 provirus, and a plasmid in which the expression of human alpha 2 interferon (HuIFN-alpha 2) was under the control of the HIV-1 long terminal repeat (LTR) (HNHIV alpha 1 cells). In both cell lines, transcription of the HIV-1 provirus was below the limits of detection, but it could be induced effectively by transfection with a HIV-1 tat-expression plasmid. In HNHIV alpha 1 cells, HuIFN-alpha 2 was induced concomitantly with HIV-1 provirus, although these cells synthesized only low levels of IFN constitutively. In contrast, infections with HSV-1 activated transcription of HIV-1 provirus only in HNHIVdt4 cells but not in HNHIV alpha 1 cells. Similarly in a transient expression assay, HSV-1 up-regulated expression of a HIV LTR-CAT (chloramphenicol acetyltransferase gene) plasmid in HNHIVdt4 but not in HNHIV alpha 1 cells. No major differences could be detected in the expression of HSV-1 immediate-early (IE) genes IE175 and IE110 (which are essential for the activation of HIV-1 LTR) in HNHIVdt4 and HNHIV alpha 1 cells to account for the inability of HSV-1 to induce HIV-1 in HNHIV alpha 1 cells. However, major differences were observed in the binding pattern of NF-kappa B-specific nuclear proteins to the enhancer region of the HIV-1 LTR: whereas binding of the 45-kDa NF-kappa B-specific nuclear protein was detected in nuclear extracts from HNHIVdt4 cells, no protein binding was seen in extracts from HNHIV alpha 1 cells. These results suggest an alternate mechanism by which IFN may alter the expression of cellular and viral genes.

Transforming growth factor-beta inhibits the antiviral action of interferons in human embryonic fibroblasts.

Transforming growth factor-beta (TGF-beta) at concentration of 10 ng/ml inhibited the development of the interferon-alpha- (IFN-alpha) or IFN-gamma-induced antiviral state in quiescent human embryonic fibroblasts. The action of the cytokines was dose-related; TGF-beta had no direct effect on the replication of either vesicular stomatitis virus (VSV) or encephalomyocarditis virus (EMCV) used as the challenge viruses in the IFN assays. We suggest that despite the fact that TGF-beta acts mainly as a "negative" growth factor, its interactions with IFNs in the antiviral assays resemble known effects of the typical "positive" peptide growth factors.

Combined action of interferons and transforming growth factor beta on the proliferation of human fibroblasts.

The effects of interferons (IFNs) and transforming growth factor-beta (TGF-beta) used alone and in combination on the multiplication of human embryonic diploid fibroblasts were studied. The experimental conditions were standardized and medium containing 2% of a single batch of fetal calf serum, which was almost minimal dose required for good the cell attachment and growth, was used. The observed effects were dose related. IFNs (types alpha, beta, or gamma) at concentration of 1 to 100 U/ml or TGF-beta at concentration of 0.1 to 1.0 ng/ml stimulated the cell multiplication by 20 to 25% when compared to the control cultures incubated without the factors. At higher doses both IFNs (10(2) to 10(5) U/ml) or TGF-beta (1 to 10 ng/ml) inhibited the proliferation of the cells. The antimitogenic action of IFNs and TGF-beta was synergistic. We suggest that IFNs as well as TGF-beta are bifunctional growth regulators although their antimitotic action dominates over their possible growth stimulatory activity. The latter action may be a secondary phenomenon due to interaction of IFNs or TGF-beta with other factors present in cell culture. We have shown that epidermal growth factor (EGF) may increase the growth stimulatory action of low doses of IFN-gamma or TGF-beta.

Bovine transforming growth factor beta. Purification and characterization.

Polypeptide growth factor isolated from bovine platelets was classified on the basis of its physico-chemical and biological properties to be transforming growth factor type beta (TGF beta). This growth factor was completely purified from platelet lysate by acid ethanol extraction, Bio-Gel P-60 filtration, ion-exchange chromatography on CM-Sephadex followed by two successive gel filtrations on Bio-Gel P-10 and Sephadex G-100. After the last gel filtration over 35,000-fold purified TGF beta was obtained. The purity of the preparation was confirmed by SDS-polyacrylamide gel electrophoresis, which revealed a single protein band with Mr of 25,000-27,000. Further studies showed that native TGF beta consists of two Mr 13,000 polypeptide chains held together by disulfide bonds.

Participation of polypeptide proteinase inhibitors in proliferation of fibroblasts.

The polypeptide proteinase inhibitors (PI) isolated from Cucurbitaceae plants, bovine pancreas, lima bean kale seeds and some other sources were assayed in mouse, rat and human cell cultures for possible mitogenic effects. PI stimulated the proliferation of the spare cultures of Balb/c 3T3 and NRK-49F fibroblasts pretreated with trypsin. PI had no effect on [3H]thymidine incorporation by the monolayer cultures of the mouse or rat cells. Also certain human cell lines were unresponsive to PI. The results suggest that PI have growth factor-like activities which are cell and/or species specific.

Interactions of interferons and transforming growth factors during clonal growth of mouse or human cells in soft agar and in mice.

The mouse fibroblast-like transformed cell line C-243 adapted to growth in suspension was used as a source of virus-induced interferons (MulFN-alpha, beta), and spontaneously produced active growth factors. These factors were purified from C-243 cells grown as tumors in BALB/c mice, and had properties identical to those of TGF-alpha or TGF-beta isolated by others from different tissues. Exogenous TGF-alpha, beta stimulated colony formation by C-243 cells in soft agar, whereas MulFN-alpha, beta inhibited it. Clonal growth of human lung adenocarcinoma A549 cells in soft agar was inhibited as well by human interferons (types alpha, beta, or gamma) as by TGF-beta. Inhibition was dose-related. Pure EGF, which is an analogue of TGF-alpha, diminished the antiproliferative activity of interferons alpha, beta, and gamma in A549 cells. On the other hand, the anti-mitogenic action of IFN-beta and TGF-beta was clearly synergistic. In mice bearing C-243 cell tumors, TGF-alpha, beta stimulated growth, whereas MulFN-alpha, beta inhibited it. Stimulation of tumor growth was also observed after administration of anti-IFN serum that could neutralize endogenous IFN-alpha, beta. The simultaneous administration of MulFN-alpha, beta and TGF-alpha, beta diminished anti-tumor effects of IFN in mice. Our results suggest that both TGFs and IFNs are autocrine, positive or negative growth factors modulating the rate of proliferation and the neoplastic behavior of the cells. The final effects depend on the target-cell sensitivity and on the relative concentration of the various hormone-like factors. Cancer cells overstimulated by TGF-alpha, beta or by EGF may not respond to IFNs.