Regulation of tamoxifen sensitivity by a PAK1-EBP1 signalling pathway in breast cancer.

BACKGROUND: EBP1, an ErbB3-binding protein, sensitises breast cancer cells to tamoxifen in part by decreasing ErbB2 protein levels. The p21-regulated serine/threonine kinase PAK1, implicated in tamoxifen resistance, phosphorylates EBP1 in vitro and in vivo at T261. Phosphorylation of EBP1 at this site induces tamoxifen resistance. We thus postulated that inhibition of PAK1 activity, by restoring EBP1 function, could ameliorate the hormone refractory phenotype of ErbB2-overexpressing breast cancer cells. METHODS: Effects of EBP1 on ErbB2 levels were measured by western blotting. Effects of EBP1 and IPA-3 on tamoxifen sensitivity were measured using a tetrazolium based cell viability assay. RESULTS: Transient transfection studies indicated that an EBP1 T261E mutant, which mimics EPB1 phosphorylated by PAK1, increased ErbB2 protein levels. An EBP1 T261A mutant, unable to be phosphorylated by PAK1, ameliorated PAK1-induced tamoxifen resistance, suggesting that phosphorylation of EBP1 by PAK1 contributes to tamoxifen resistance. We then tested if pharmacological inhibition of PAK1 activity might render hormone resistant cells, which endogenously overexpress PAK1, tamoxifen sensitive. IPA-3, a specific small MW PAK1 inhibitor, sensitised cells to tamoxifen only when EBP1 was ectopically expressed. IPA had no effect on tamoxifen resistance in T47D cells in which EBP1 protein had been ablated by shRNA. The IPA-induced increase in tamoxifen sensitivity was accompanied by a decrease in ErbB2 levels only in EBP1-overexpressing cells. CONCLUSION: These studies suggest that phosphorylation of EBP1 may be one mechanism of PAK1-induced hormone resistance and that PAK1 inhibitors may be useful in cells in which EBP1 is overexpressed.

Side-population cells in luminal-type breast cancer have tumour-initiating cell properties, and are regulated by HER2 expression and signalling.

BACKGROUND: The expression of side-population (SP) cells and their relation to tumour-initiating cells (T-ICs) have been insufficiently studied in breast cancer (BC). We therefore evaluated primary cell cultures derived from patients and a panel of human BC cell lines with luminal- or basal-molecular signatures for the presence of SP and BC stem cell markers. METHODS: The SPs from luminal-type BC were analysed for BC T-IC characteristics, including human epidermal growth factor receptor 2 (HER2), ERalpha, IGFBP7 expression and their ability to initiate tumours in non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice. Pharmacological modulators were used to assess the effects of HER2 signalling and breast cancer-resistance protein (BCRP) expression on SPs. RESULTS: The SP was more prevalent in the luminal subtype of BC compared with the basal subtype. HER2 expression was significantly correlated with the occurrence of an SP (r(2)=0.75, P=0.0003). Disappearance of SP in the presence of Ko143, a specific inhibitor of the ATP-binding cassette transporter BCRP, suggests that BCRP is the predominant transporter expressed in this population. The SP also decreased in the presence of HER2 signalling inhibitors AG825 or trastuzumab, strengthening the notion that HER2 contributed to the SP phenotype, likely through downstream AKT signalling. The SP cells from luminal-type MCF-7 cells with enforced expression of HER2, and primary cells with luminal-like properties from a BC patient, displayed enrichment in cells capable of repopulating tumours in NOD/SCID mice. Engraftment of SP cells was inhibited by pretreatment with AG825 or by in vivo treatment with trastuzumab. INTERPRETATION: Our findings indicate an important role of HER2 in regulating SP and hence T-ICs in BC, which may account for the poor responsiveness of HER2-positive BCs to chemotherapy, as well as their aggressiveness.

Primary bioassay of human tumor stem cells.

A simple method has been developed to support human tumor stem cell colony growth in soft agar. The technique appears suitable for culture of a variety of neoplasms of differing histopathology. Tumor stem cell colonies arising from different types of cancer have differing growth characteristics and colony morphology. This bioassay should be suitable for clinical studies of effects of anticancer drugs or irradiation on human tumor stem cells.

Inhibition of B-lymphocyte clonal proliferation by spleen cells from plasmacytoma-bearing mice.

This study demonstrated that spleen cells of inbred BALB/c mice with plasmacytomas inhibited proliferation of the B-lymphocyte colony-forming cells that were precursors of immunoglobulin-producing cells. The number of B-lymphocyte colonies/10(5) nucleated spleen cells was depressed in mice with plasmacytomas. The degree of suppression correlated with the size of the tumor. Spleen cells from tumor-bearing mice suppressed the formation of B-lymphocyte colonies by normal BALB/c spleen cells. Treatment of the myeloma spleen cell suspensions with anti-immunoglobulins, anti-theta, or 1,500 rads did not prevent the suppression. Spleen cells from tumor-bearing mice depleted of adherent cells by passage over Sephadex G-10 columns or adherence to plastic dishes did not retain their suppressive activity. Adherent spleen cells inhibited B-lymphocyte colony formation when they were either in direct contact with normal spleen cells or separated from them by a layer of agar. These studies suggested that the immune suppression seen in plasmacytoma-bearing mice occurred partially at the level of the B-lymphocyte progenitor cell and that this suppression was mediated by an adherent mononuclear cell.

Effect of epidermal growth factor on proliferation of human tumor cells in soft agar.

The effect of epidermal growth factor (EGF) and fibroblast feeder layers on the proliferation of human tumor cells in soft agar was examined. The addition of EGF to medium supporting growth of tumor cells significantly increased (P less than or equal to 0.02) the number of colonies grown from the cells of 36 to 58 patients (62%) with a variety of neoplasms. The increase in the number of colonies was dependent on the concentration of EGF and was maximal at a concentration of 50 ng EGF/ml. The addition of glucocorticoids did not potentiate the effect of EGF. Lethally irradiated fibroblast feeder layers alone only slightly increased the number of colonies. However, the addition of fibroblasts to cultures significantly increased the mitogenic effect of EGF. The results suggest that a proportion of epithelial-derived tumors retain responsiveness in vitro to physiologic growth regulators such as EGF.

Effect of enzymatic disaggregation on proliferation of human tumor cells in soft agar.

Cell suspensions were prepared by either mechanical or enzymatic disaggregation methods from biopsy specimens from 54 patients with various tumors. The biologic activities of cells derived from the two suspensions were then examined. Biopsy specimens of solid tumors were minced, and one-half of each specimen was further processed by being teased with needles, whereas the other half was exposed to a combination of collagenase, hyaluronidase, and DNase. The enzymatic disaggregation method yielded fewer cells per gram of tissue than the mechanical method. However, the percentage of dye-excluding cells was increased by the enzymatic procedure in 93% of the cases. Cells obtained by enzymatic means also had higher cloning efficiencies than those obtained by mincing. The histologic types of cells present in the initial cell suspensions were the same for cells obtained by the enzymatic or mechanical disaggregation methods. The number of colonies obtained was linearly related to the number of cells plated in both cases. The tritiated thymidine suicide indices (estimates of the percentage of cells in the S-phase of the cell cycle) were the same for the two cell populations obtained by the two methods. The results indicate that cells obtained from solid tumors by enzymatic dissociation methods did not differ significantly from cells obtained by the more conventional mechanical techniques. However, cell viabilities and cloning efficiencies were significantly improved by the enzymatic technique.

Separation on Percoll density gradients of cells derived from malignant ascites of mice.

In an attempt to separate malignant from normal and reactive stromal cells, we fractionated ascites cells from BALB/c mice bearing a transplantable myeloma (MPC-11) by isopyknic centrifugation in continuous density gradients of povidone-coated silica gels (Percoll). Cells from different fractions were then analyzed by morphologic and immunologic criteria. The ability of cells from the different fractions to form colonies in soft agar and to produce tumors in BALB/c mice was also examined. Although most fractions contained morphologically identifiable plasma cells, colony-forming cells (CFC), derived from multiply passaged tumors, separated in a sharp peak at 1.072 g/ml. CFC peaked at 1.078-1.082 g/ml for tumors passed less than three times and were invariably markedly depleted from the low-density portions of the gradients. Cells recovered from different fractions of the gradients were cultured in soft agar and inoculated sc into syngeneic mice. In these experiments, a highly significant correlation was observed between the ability of cells to form colonies in soft agar and to form tumors in vivo. This correlation suggests that CFC and tumorigenic cells have similar distributions.

O-phospho-L-tyrosine inhibits cellular growth by activating protein tyrosine phosphatases.

O-Phospho-L-tyrosine (P-Tyr), a substrate for a wide range of protein tyrosine phosphatases, inhibited growth of human renal and breast carcinoma cells. Growth was blocked in the S phase of the cell cycle. A decrease in the amount of cyclin proteins A and B was also observed. P-Tyr incubation led to activation of cellular protein tyrosine phosphatases resulting in the inhibition of tyrosine phosphorylation of epidermal growth factor receptor as well as of p34cdc2. P-Tyr synergistically sensitized the renal carcinoma ACHN cells to killing by the chemotherapeutic agents doxorubicin and etoposide. These growth inhibitory properties of P-Tyr in vitro suggest its possible use as an anticancer agent.

Stimulation of anchorage-independent growth of human tumor cells by interleukin 1.

Human peripheral blood monocytes and tumor-associated macrophages release a factor that enhances the clonal growth of a human epithelial tumor cell line (SW-13) in soft agar. We now demonstrate that purified interleukin 1 (IL-1) may account for part of this colony-stimulating activity. Purified IL-1 (0.5 to 8 units/ml) was added to SW-13 cells cultured in soft agar. IL-1 increased colony growth in a dose-dependent manner and did not inhibit colony formation at the highest doses tested. Other purified human monocyte products (alpha-interferon, tumor necrosis factor, transforming growth factor beta, fibronectin) did not stimulate colony growth. Antibody to IL-1 only partially inhibited the ability of monocyte-conditioned medium to stimulate SW-13 colony growth. This antibody did, however, completely inhibit the ability of purified IL-1 to support the growth of SW-13 colonies in soft agar. IL-1 increased growth of quiescent SW-13 cells cultured in monolayers as assessed by tritiated thymidine incorporation assays. The results of this study indicate that IL-1 can enhance clonogenic growth of an epithelial cell line in soft agar. However, other uncharacterized activities in monocyte conditioned medium also promote colony growth. These studies add to an increasing body of evidence indicating that inflammatory products play a role in maintaining the transformed phenotype.

Cytotoxicity of human beta-interferon for differentiating leukemic HL-60 cells.

We examined the effect of human interferon (HuIFN) -alpha and -beta on the proliferation and differentiation induced by dimethyl sulfoxide (DMSO) of HL-60 human promyelocytic leukemia cells into mature granulocytes. Neither HuIFN-alpha nor -beta, alone, from 1 to 1000 IU/ml, nor the homologous mock HuIFN preparations affected HL-60 cell differentiation or proliferation. Whereas the combination of HuIFN-alpha (10 to 1000 IU/ml) with DMSO also did not affect the proliferation or differentiation of HL-60 cells, the addition of HuIFN-beta (1000 IU/ml) and DMSO (1.25%) to growing cultures reduced cell viability as much as 14% of that observed for cells treated with DMSO alone or to 4% of that observed for either untreated cells or those treated with HuIFN-beta alone. The cytotoxic effect declined with decreasing concentrations of HuIFN-beta. The cytotoxic effect of DMSO and HuIFN-beta was exerted only as cells began to differentiate. Removal of HuIFN-beta at Day 2 did not reverse the cytotoxic effect, and addition of HuIFN-beta at Day 2 did not inhibit cell proliferation. Addition of HuIFN-beta to postmitotic cells on Day 4 after DMSO treatment did not affect proliferation but did slow differentiation. The cytotoxic and antidifferentiative effects of naturally produced HuIFN-beta were confirmed with highly purified recombinant HuIFN-beta. Undifferentiated HL-60 cells were resistant to the antiviral effects of HuIFN-beta, requiring 4 to 6 times the concentration to protect 50% of the cells against vesicular stomatitis virus as that needed to produce a cytotoxic or antidifferentiative effect. The profoundly cytotoxic effects of HuIFN-beta reported here may provide a model to study this interferon in combination with inducers of leukemic cell differentiation as a possible strategy in cancer therapy.

Induction of differentiation of human promyelocytic leukemia cells (HL60) by metabolites of hexamethylene bisacetamide.

We studied the ability of five metabolites of hexamethylene bisacetamide (HMBA), which we had previously identified in patient urine, to induce differentiation or to influence differentiation induced by HMBA of a human promyelocytic cell line. Differentiation of HL60 cells was quantified by morphological changes and by the ability to reduce nitroblue tetrazolium. N-Acetyl-1,6-diaminohexane (NADAH), the deacetylated, first metabolite of HMBA, was a more potent inducer of HL60 differentiation than was HMBA. NADAH produced 20-30% differentiation at 0.25 mM and 30-40% differentiation at 0.5 mM. NADAH (1 mM) induced 2-3-fold more differentiation than did 1 mM HMBA. HL60 differentiation, induced by various combinations of HMBA and NADAH, reflected a combined effect of the two compounds. In contrast, 1,6-diaminohexane, at 0.5-5 mM, failed to induce HL60 differentiation. Similarly, 0.5-5 mM 6-acetamidohexanoic acid, the major metabolite of HMBA, and 6-aminohexanoic acid failed to induce differentiation of HL60 cells. However, 6-acetamidohexanoic acid, when combined with HMBA or NADAH at various concentrations and ratios, enhanced the differentiation of HL60 cells induced by these two compounds. This enhancement was most apparent with addition of 0.50-3.0 mM 6-acetamidohexanoic acid to HL60 cells incubated with 1.0-3.0 mM HMBA or 0.25-1.0 mM NADAH. 6-Aminohexanoic acid similarly enhanced HMBA-induced differentiation of HL60 cells. These in vitro results have implications in terms of the clinical application of HMBA and interpretation of the results of clinical trials performed to date and may provide some insight into the mechanism of HMBA-induced cellular differentiation.

Isolation and characterization of a monoclonal antibody specific for epithelial cells.

Monoclonal antibodies were generated to antigens on human foreskin keratinocytes to identify epithelial-specific molecules. Spleen cells from BALB/c mice, immunized with membrane preparations from primary explants of foreskin epithelial cells, were fused with the NS-1 mouse myeloma line. Hybridoma supernatants were screened for the desired immunological reactivity using enzyme-linked immunosorbant binding assays. Hybridomas secreting antibodies reacting with epithelial cells, but not fibroblasts or lymphocytes, were cloned by limiting dilution, and two stable clones producing immunoglobulin M K antibodies were selected for study. Evaluation of fixed paraffin-embedded human tissue by an indirect immunoperoxidase technique revealed that the antibodies bound most strongly to normal stratified squamous and transitional epithelium, and squamous and transitional cell carcinomas. Antibodies from the cloned hybridomas also reacted with primary cell cultures of foreskin keratinocytes, pulmonary epithelium, fetal liver, and amnion cells, but not with primary cultures of nonepithelial cells. Further testing by enzyme-linked immunosorbent assays revealed that the antibodies reacted with some long-term cell lines derived from epithelial tumors. Nonepithelial cell lines were not stained by the antibodies. Indirect immunofluorescent studies indicated that staining was confined to the cell surface. These antibodies may prove useful in studies of differentiation markers of human epithelial cells.

A microtiter enzyme-linked immunosorbent assay for protein tyrosine phosphatase.

We report the development of an enzyme-linked immunosorbent assay (ELISA) for protein tyrosine phosphatases (PTPases). PTPase activity, was monitored by quantitating the disappearance of O-phospho-L-tyrosine (P-Tyr) in an ELISA system using antigen capture followed by double antibody labelling. PTPase activity of agarose conjugated PTP-1B was demonstrated using the ELISA system. PTPase activity was sensitive to both PTB-1B concentrations and time of incubation. 1 mU of PTPase activity was defined as that amount of enzyme producing a rate of loss of 0.01 absorbance units/minute with a specific activity of 150 pmol P-Tyr/min per micrograms protein based on the unit of PTPase activity from the conventional assay system. The PTP-1B activity was shown by the ELISA system to be completely inhibitable by Poly (Glu,Tyr)4:1 at 100 micrograms/ml. We used the ELISA system to detect PTPase activity in lysates of cultured cells. The PTPase activity of cell lysates of MDA-MB 468 breast carcinoma cells as obtained by the ELISA were compared with those obtained by a standard 32P(i) release assay using radio-labelled Raytide as PTPase substrate. The decrease in P-Tyr concentration was dependent on the time of incubation with the lysate and on lysate concentration and compared well with the release of 32P(i) in the radioactive assay system. Orthovanadate as well as heat denaturation inhibited the PTPase activity of the cell lysates in both the assay systems. The assay presented here is a simple immunological system capable of measuring activity of purified PTPases as well as PTPase levels in cell and tissue extracts.

Monocyte-derived growth factors for human tumor clonogenic cells.

Human peripheral blood monocytes secrete a soluble factor that enhances the ability of human epithelial tumor cell lines to clone in soft agar. Monocytes increased colony growth in a concentration-dependent manner, although inhibition of cell growth was observed in the presence of high concentrations of monocytes. Addition of indomethacin to monocytes did not abrogate this inhibition. Exposure of monocytes to endotoxin increased their ability to secrete stimulatory factors. Nonadherent lymphocytes were unable to support colony growth. Conditioned media from unstimulated monocytes also increased colony growth. Growth-promoting activity was detected in the media within the first 24 hr of culture, reaching a peak at 72 hr. Activity was not observed in monocyte lysates. Monocytes released this activity when cultured in the presence of both serum-free and serum-containing media. The activity was nondialyzable, relatively heat stable, and failed to adhere to CM-Sephadex. The demonstration of a monocyte-derived factor that enhances growth of epithelial tumor colonies supports findings indicating that inflammatory products may enhance tumor cell growth in vitro.

Amelioration of azidothymidine-induced erythroid toxicity by hemin and stem cell factor in immune-suppressed mice.

Recombinant cytokines such as stem cell factor (SCF) are currently being tested for the ability to ameliorate 3'azido-3'deoxythymidine (AZT)-induced anemia in AIDS patients. Recently, we showed that SCF greatly increased burst-forming units-erythroid (BFU-E) but failed to increase hematocrits of AZT-treated immune-deficient (MAIDS) mice. We reasoned that hemin, previously shown to both enhance BFU-E proliferation and accelerate erythroid maturation, might bring about differentiation of this large SCF-induced pool of BFU-E and further protect BFU-E from AZT's toxic effect. We therefore studied, in vitro, the effect of combinations of hemin and SCF on growth of BFU-E from MAIDS mice. Hemin, at concentrations of 10 to 100 microM, ameliorated the growth-inhibitory effect of AZT. 50 microM hemin increased the ED50 of AZT from 1 x 10(-7) M to 1.7 x 10(-6) M. SCF also ameliorated AZT-induced toxicity, but to a lesser extent. SCF and hemin increased the number of BFU-E colonies observed in the presence of AZT in an additive fashion. The resistance of BFU-E to AZT's cytotoxic effect was greater in cultures receiving hemin and SCF together than in cultures receiving SCF or hemin alone. Zinc and tin protoporphyrins (Zn and Sn PP) increased the numbers of BFU-E observed. However, neither zinc nor tin protoporphyrins increased the ED50 of AZT. Combinations of SCF and hemin may prove useful in ameliorating AZT toxicity in both immune-suppressed mice and human immunodeficiency virus (HIV)-infected patients.

Time-dependent changes induced by azidothymidine in erythroid progenitor colonies in immunodeficient mice.

The effect of azidothymidine (AZT) on erythropoiesis in C57BL/6 mice made immunodeficient by infection with LP-BM5 murine leukemia virus (MuLV) was examined. Earlier work from our laboratory indicated that long-term treatment of LP-BM5 MuLV-infected mice with AZT induced peripheral anemia but increased the number of splenic and bone marrow erythroid burst-forming units (BFUe). In contrast, other workers have demonstrated that short-term intensive AZT treatment decreases bone marrow BFUe of normal mice. The purpose of the present study was to determine the effects of short-term oral AZT treatment in immune deficient animals. LP-BM5 MuLV-infected and normal mice were given 0, 1, and 2.5 mg/ml of AZT in their drinking water. Mice were killed after 2, 4, 8, 15, and 30 days of AZT treatment. The hematocrits of all AZT-treated mice decreased in a dose- and time-dependent fashion. AZT treatment decreased the absolute numbers of circulating reticulocytes in both normal and infected mice after 4 days of treatment. In contrast, the percentage of bone marrow early erythroblasts was increased in both normal and infected animals after 4 days of treatment. AZT at both doses decreased the number of BFUe per femur in both infected and normal mice after 2, 4, and 8 days. However, after 15 days the number of bone marrow BFUe increased. In spleen, the numbers of BFUe were increased only with high-dose AZT in both normal and infected mice at all time points, although the increases were more dramatic in infected mice. Our results indicate that the effect of AZT on bone marrow BFUe is time dependent, with inhibition being observed only at early time points. These results further demonstrate the complex effects of AZT on erythropoiesis in vivo.

Effects of in vivo administration of stem cell factor on thrombopoiesis in normal and immunodeficient mice.

Thrombocytopenia is an important clinical problem for many acquired immunodeficiency syndrome (AIDS) patients. Recently, the utility of recombinant cytokines in alleviating the hematopoietic complications of AIDS and AIDS therapy has been evaluated. The newly cloned cytokine stem cell factor (SCF) has been demonstrated to be a potent regulator of hematopoietic progenitor cell proliferation. Therefore, we evaluated the ability of SCF to alleviate thrombocytopenia caused by infection with LP-BM5 murine leukemia virus (mLV) in a murine model of AIDS (MAIDS). In addition, we evaluated the effects of SCF on previously demonstrated azidothymidine (AZT)-induced elevations of platelet counts. SCF was administered to normal or LP-BMS-infected C57BL/6 mice in combination with oral AZT for up to 1 month and effects on platelet, megakaryocyte (MK), and megakaryocyte colony-forming cell (CFU-MK) numbers were evaluated. SCF alone significantly increased the number of circulating platelets in thrombocytopenic MAIDS mice by 53%. SCF also significantly elevated platelet levels by 29% in normal mice. AZT elevated platelet counts 100% in normal and 50% in MAIDS mice. AZT and SCF increased platelet counts in an additive manner. SCF alone was a potent inducer of splenic CFU-MK in both MAIDS and normal mice, increasing splenic CFU-MK 13- to 15-fold at day 15 as compared with untreated controls. By day 30, however, the numbers of splenic CFU-MK had returned to control levels. In infected mice, AZT alone increased the number of splenic CFU-MK. SCF administered to AZT-treated MAIDS mice did not further enhance these increases. In contrast, in normal mice, AZT decreased splenic CFU-MK numbers. In AZT-treated mice, SCF enhanced the numbers of splenic CFU-MK 90-fold at day 8. In MAIDS mice, the number of bone marrow CFU-MK was significantly increased by SCF treatment at all time points. SCF significantly affected the total number of femoral CFU-MK in AZT-treated mice only at day 15. In normal mice, SCF or SCF and AZT in combination increased the total number of bone marrow CFU-MK five-fold at day 8, but failed to induce changes in the total number of femoral CFU-MK after that. These results indicate that SCF elevates platelet levels in both thrombocytopenic MAIDS and normal mice and profoundly affects CFU-MK proliferation. Combinations of SCF and AZT may be further explored to enhance the therapeutic effectiveness of these two drugs in alleviating thrombocytopenia.

Recombinant human interferon alpha enhancement of retinoic-acid-induced differentiation of HL-60 cells.

We have previously demonstrated that a combination of interferon beta and a differentiation agent, dimethyl sulfoxide (DMSO), is cytotoxic for HL-60 cells, a human promyelocytic leukemic cell line. We now report that a combination of recombinant interferon alpha (Intron; Schering) and retinoic acid is synergistically cytostatic for HL-60 cells. Retinoic acid (RA) induced the differentiation of HL-60 cells into granulocytes. Interferon (IFN) alone at 1-1000 IU/ml had no effect on either differentiation or proliferation of HL-60 cells. The addition of 1000 IU/ml of IFN and 10(-7) M RA at the initiation of culture reduced the number of viable cells to 28% of that observed for cells treated with RA alone. The decreased number of cells was a result of decreased cellular proliferation, rather than of a cytotoxic effect of the combination. IFN-RA-treated cells differentiated more rapidly than cells treated with RA alone. In addition, the final percentage of mature cells was increased at day 7 in IFN-RA-treated cultures, as compared with RA-treated cells. Simultaneous treatment of the cells with IFN and RA decreased the concentration of RA needed to induce differentiation or to exert a cytostatic effect. Significant changes in the nuclear structure of RA-treated HL-60 cells after 24 h have been reported. Cells were pulsed with RA for 24 h, washed, and IFN added. At day 7, cell growth was inhibited to the same extent as that of cells continuously exposed to IFN-RA. However, while 70% of the continuously exposed cell differentiated, cells pulsed with RA and subsequently treated with IFN did not differentiate. The results of this investigation further support our findings that combinations of IFN and inducers of differentiation may be of importance in the treatment of leukemia.

3'-Azido-3'-deoxythymidine ameliorates the thrombocytopenia observed in a murine model of AIDS.

3'-Azido-3'-deoxythymidine (AZT) is used in the management of acquired immunodeficiency syndrome (AIDS) and AIDS-related complex (ARC). The myelotoxic actions of AZT are well known but its effect on platelets is not clear. We studied the effect of AZT at 1 and 2.5 mg/ml in drinking water on platelets in a murine model of AIDS. Three stages of the disease were examined, as determined by the serum IgM levels and other physical features. As early as 15 days after the initiation of drug treatment, AZT was found to significantly increase platelet production. To ascertain that this activity was authentic, a further study was carried out using uninfected mice. Mice were given AZT at both doses for 15 and 30 days. All mice on AZT had significantly increased numbers of platelets. These increases were dose and time dependent. AZT is therefore a potent inducer of thrombocytosis and may be a potential candidate in the treatment of thrombocytopenia in AIDS.

Modulation of B-cell colony growth by T-cell subsets.

The ability of T-cell subsets to modulate the growth of B-cell colonies derived from human peripheral blood mononuclear cells (PB-MNC) was analyzed. B-cell-enriched populations (BCE), isolated by depletion of E-rosetting cells, were placed in the upper agar layer of a double-layer agar system. The lower layer contained the mitogen Staph Protein A (5-10 micrograms/ml). Colonies of B cells (100-500/4 X 10(5) cells) were observed in cultures containing BCE, partially depleted of T cells by a single cycle of E-rosetting. Rigorous depletion of T-lymphocytes from BCE by an anti-T-cell antibody and C' decreased colony numbers to approximately 25% of control values. Addition of autologous T cells to underlayers restored B-cell colony growth in a dose-dependent fashion. OKT4 cells were more effective than OKT8 cells in enhancing colony responses. OKT8 cells, however, did not suppress B-cell colony growth. The ability of T cells to enhance proliferation of B cells was unaffected by irradiation. These data indicate that T cells amplify proliferation of human B-cell colonies and that different subsets of T cells vary in their ability to support B-cell colony growth.