Effect of isothermal radiofrequency radiation on cytolytic T lymphocytes.

Previous in vitro studies provide evidence that RF electromagnetic radiation modulates proliferation of human glioma, lymphocytes, and other cell types. The mechanism of RF radiation cell proliferation modulation, as well as mechanisms for effects on other cell physiologic endpoints, are not well understood. To obtain insight regarding interaction mechanisms, we investigated effects of RF radiation exposure on interleukin 2 (IL-2) -dependent proliferation of cytolytic T lymphocytes (CTLL-2). After exposure to RF radiation in the presence or absence of IL-2 cells were cultured at various physiological concentrations of IL-2. Treatment effects on CTLL-2 proliferation were determined by tritiated thymidine incorporation immediately or 24 h after exposure. Exposure to 2450 MHz RIF radiation at specific absorption rates (SARs) of greater than 25 W/kg (induced E-field strength 98.4 V/m) induced a consistent, statistically significant reduction in CTLL-2 proliferation, especially at low IL-2 concentrations. At lower SARs, 2450 MHz exposure increased CTLL-2 proliferation immediately after exposure but reduced 24 h postexposure proliferation. RF radiation effects depended on the mitotic state of the cells at the time of exposure. Comparison of the effects of temperature elevation and RF radiation indicated significant qualitative and quantitative differences.

Effects of the protein kinase C stimulant bryostatin 1 on the proliferation and colony formation of irradiated human T-lymphocytes.

The protein kinase C stimulant bryostatin 1 (Bryo) was used in examining human peripheral blood T-lymphocyte radiosensitivities in proliferation assays. Bryo was similar to PMA in inducing T-cell proliferation by the CD3, CD28 and CD69 pathways. No difference in radiosensitivities was observed in T-cells stimulated by the three independent surface antigen-mediated activation pathways. CD3 was chosen as the second signal for comparing the potencies of the three different first signals Bryo, phorbol 12-myristate, 13-acetate (PMA), and interleukin 2 (IL-2) in stimulating T-cell proliferation and in maintaining this response after radiation. Though there were radioresponse differences among various individuals, the irradiated lymphocytes consistently showed significantly greater proliferation when treated with Bryo or PMA than with IL-2 (p < 0.05- < 0.005). No difference in proliferative responses was observed in T-cells irradiated between 4 h before and 15 h after the addition of stimulants. Colony forming assays showed higher colony survival for irradiated T-cells stimulated with Bryo than with PMA. These results support the important role of protein kinase C in T-cell radiation responses, and suggest a potential role for Bryo in enhancing T-lymphocyte survival during radiation therapy.

Expansion and tumour specific cytokine secretion of bryostatin-activated T-cells from cryopreserved axillary lymph nodes of breast cancer patients.

Current adoptive immunotherapy strategies in cancer patients require large numbers of activated T-cells and are limited by the availability of autologous tumour. We describe a novel method of T-cell activation that produced relatively rapid, high-fold expansion of stored, frozen lymphocytes obtained from the lymph nodes of 20 breast cancer patients during axillary dissection but does not require autologous tumour. In vitro exposure of thawed cells to bryostatin-1 (B), a non-tumour promoting protein kinase C activator and ionomycin (I), a calcium ionophore, at day 0 followed by culture in low dose interleukin-2 (IL-2 20 units ml-1) and restimulation again on day 10 results in 269-28,206 fold (geometric mean = 2254) expansion in cell numbers counted 17 days after initial stimulation. Analysis of cell surface markers revealed that B/I expanded human cells were predominantly T-cells (83-97%) and consisted of a mixture of CD8+ (46-74%) and CD4+ (4-30%) cells. B/I expanded cells did not lyse autologous tumour cells when tested in a 4-h 51Cr release assay, but murine studies reported previously have demonstrated specific and curative in vivo efficacy in MCA-105 tumour-bearing mice despite an inability to lyse autologous tumour in vitro. B/I expanded T-cells from five of six patients secreted the cytokines tumour necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) in response to co-culture with autologous tumour cells but not with irrelevant tumour. These results are analogous to findings in a murine model, in which non-cytolytic B/I expanded T-cells mediated specific, curative anti-tumour effects in vivo, and lay the groundwork for a clinical trial of this novel strategy for the adoptive immunotherapy of breast cancer patients.

Modulation of the activity of a human granulocyte-macrophage colony-stimulating factor/interleukin-3 fusion protein (pIXY 321) by the macrocyclic lactone protein kinase C activator bryostatin 1.

We have examined the effect of the macrocyclic lactone protein kinase C (PK-C) activator bryostatin 1 on the proliferative capacity and lineage commitment of CD34+ human bone marrow cells exposed to the granulocyte-macrophage colony-stimulating factor/interleukin-3 (GM-CSF/IL-3) fusion protein pIXY 321. pIXY 321 administered at a dose of 10 ng/mL was as effective as the combination of plateau concentrations of recombinant (r) IL-3 and rGM-CSF (e.g., 50 ng/mL) in stimulating the growth of day-14 committed myeloid progenitors (colony-forming units granulocyte/macrophage [CFU-GM]). In the large majority of samples tested, coadministration of 0.5 to 100 nM bryostatin 1 with either pIXY 321 or the combination of rIL-3 plus rGM-CSF led to modest but significant increases (e.g., 30 to 75%) in the number of CFU-GM, compared to administration of growth factors alone. The degree of bryostatin 1-induced potentiation, however, was considerably less than that previously observed in the case of cells exposed to either rIL-3 or rGM-CSF, where increases of 100 to 150% were regularly noted. While at least 50% of day-14 CFU-GM stimulated by either pIXY 321 or the combination of rIL-3 plus rGM-CSF were of the pure or mixed eosinophilic variety, coadministration of bryostatin 1 resulted in a dramatic inhibition of eosinophilic colonies and a corresponding increase in pure and mixed neutrophil and macrophage colonies. Although coadministration of recombinant granulocyte colony-stimulating factor (rG-CSF) or recombinant colony-stimulating factor-1 (rCSF-1) mimicked the capacity of bryostatin 1 to increase the total number of pIXY 321-induced day-14 CFU-GM, these growth factors, unlike bryostatin 1, were not capable of inhibiting eosinophilic colony formation. Furthermore, whereas addition of neutralizing antibodies to G-CSF or CSF-1 blocked the capacity of these growth factors to potentiate colony formation in the presence of pIXY 321, it did not abrogate the effect of bryostatin 1 on progenitor cell growth or lineage commitment. Finally, in contrast to its effects on committed myeloid progenitors, bryostatin 1 did not increase the growth of erythroid (burst-forming units-erythroid [BFU-E]) and multipotent (multipotent colony-forming units [CFU-GEMM]) progenitors stimulated by pIXY 321, but instead inhibited colony formation at higher concentrations (e.g., 10 to 100 nM).(ABSTRACT TRUNCATED AT 400 WORDS)

gamma-Interferon plays a key role in T-cell-induced tumor regression.

Recent studies have demonstrated that noncytolytic T-cells can mediate regression of murine tumors. In this report, we demonstrate that MCA-105 tumor-draining lymph node cells (DLN) activated with the protein kinase C activator, bryostatin 1, plus a calcium ionophore are capable of inducing specific tumor regression in vivo when adoptively transferred to mice with established metastases. However, these activated DLN cells lack in vitro cytotoxicity against autologous tumor. Antibody against gamma-interferon (IFN-gamma) markedly inhibited the therapeutic efficacy of these activated DLN cells. Anti-tumor necrosis factor produced a statistically significant but weaker inhibition of tumor regression. IFN-gamma, but not tumor necrosis factor alpha, could be shown to be secreted by activated DLN cells in vitro in response to specific tumor. Secretion of IFN-gamma was primarily a function of CD8+ T-cells. IFN-gamma was not directly cytotoxic to sarcoma cells in vitro. Moreover, tumor cells incubated with IFN-gamma were not more susceptible to lysis by activated DLN cells. However, recombinant murine IFN-gamma had a significant antiproliferative effect against MCA-105 tumor cells when tested in a [3H]thymidine uptake assay. Similarly, supernatants obtained from DLN/autologous tumor cocultures markedly inhibited MCA-105 proliferation; this antiproliferative effect was abrogated by the addition of anti-IFN-gamma antibody to the cultures. These results suggest that secretion of IFN-gamma by adoptively transferred DLN cells plays an essential role in tumor rejection. The dominant effect of IFN-gamma may be its demonstrated antiproliferative activity.

Bryostatin 1 modulates the proliferation and lineage commitment of human myeloid progenitor cells exposed to recombinant interleukin-3 and recombinant granulocyte-macrophage colony-stimulating factor.

The activity of protein kinase C (PK-C) has been implicated in the regulation of the growth and differentiation of both normal and neoplastic hematopoietic cells. We have examined the effects of the PK-C-activating agents phorbol 12,13-dibutyrate (PDBu), mezerein, and bryostatin 1 on the proliferation and lineage commitment of CD34+ human myeloid progenitor cells stimulated by recombinant interleukin-3 (rIL-3) and/or recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF). Although each of the PK-C activators administered alone induced no colony formation, coadministration of these agents with plateau concentrations of each cytokine (eg, 50 ng/mL) increased the number of day 14 granulocyte-macrophage colony-forming units by 100% to 150%. The number of pure and mixed neutrophil and macrophage colonies was substantially enhanced in the presence of PK-C activators, whereas the percentage and, in most cases, the absolute number of eosinophilic colonies was significantly reduced. The inhibition of eosinophilic colony formation was not overcome by the addition of rIL-5. Although addition of bryostatin 1 24 hours before rIL-3 abrogated the increase in total colony formation observed with simultaneous administration of factors, the inhibition of eosinophilic colonies and the increase in neutrophil/macrophage colonies persisted under these conditions. The addition of bryostatin 1 for up to 144 hours after rIL-3 continued to potentiate total colony formation, whereas the inhibition of eosinophilic commitment was lost after 120 hours. Together, these results suggest that pharmacologic interventions at the level of PK-C may regulate both the proliferation as well as the lineage commitment of human hematopoietic progenitors exposed to rGM-CSF and rIL-3.

Bryostatin 1 activates T cells that have antitumor activity.

Several strategies have been used to stimulate the growth of tumor-specific T cells in place of tumor antigen. One approach is to use pharmacologic agents to activate the second messenger pathways of T-cell activation. In the present study, we examined the ability of the protein kinase C activator bryostatin 1 (B) plus the calcium ionophore ionomycin (I) to stimulate the growth of lymphocytes obtained from the axillary lymph nodes (DLN) draining a progressively growing intradermal plasmacytoma tumor. Draining lymph node cells were initially cultured with autologous tumor cells and 20 U/ml of interleukin-2 (IL-2) for 7 days. The lymphocytes were then incubated with various concentrations of bryostatin 1 plus 1 microM ionomycin and cultured for an additional 14 days in IL-2. DLN cells initially cultured with autologous tumor and then restimulated with 5 nM bryostatin 1 and 1 microM ionomycin exhibited marked in vitro proliferation and 15-fold expansion of cell numbers over 2 weeks. The cells expanded with B/I were predominantly CD8+ T cells and retained specific in vitro cytotoxicity against autologous tumor. When adoptively transferred to mice with established liver metastases, DLN cells restimulated with B/I-mediated specific tumor regression.

Activation of CD8+ murine T cells from tumor-draining lymph nodes by phorbol dibutyrate plus calcium ionophore.

When lymphocytes from the lymph nodes draining the site of a progressively growing MCA-105 sarcoma are stimulated in vitro with autologous tumor and low-dose interleukin-2 (IL-2), they will grow and develop the ability to lyse autologous tumor cells in vitro; these lymphocytes can also eradicate tumor metastases in vivo. Phorbol esters and calcium ionophores activate signal transduction pathways in T cells and mimic the events triggered by antigen binding. We therefore sought to determine whether large numbers of MCA-105 tumor-specific, therapeutically active T cells could be obtained from MCA-105 draining lymph nodes (DLNs) following a brief exposure to phorbol dibutyrate (PDBu) and ionomycin (Io). DLN cells primarily stimulated with autologous tumor, followed by a secondary stimulation with PDBu-Io and cultured in 20 U/ml IL-2, demonstrated marked expansion of cell numbers during 3 weeks in culture, had moderate cytolytic activity [37% at effector:target ratio (E:T) = 80:1], and were all CD8+ T cells. In contrast, DLN cells stimulated primarily with PDBu-Io and cultured in 20 U/ml IL-2 demonstrated at least 8-10-fold greater growth than antigen-stimulated DLN cells during 3 weeks, were moderately cytolytic (31% at E:T = 80:1), and were a mixed population of CD8+ and CD4+ T lymphocytes. DLN cells that were expanded by either protocol, like cells stimulated repeatedly in vitro with tumor cells, could eliminate MCA-105 pulmonary metastases when given with IL-2 in an adoptive immunotherapy model. DLN cells stimulated primarily with PDBu-Io completely eradicated MCA-105 metastases but had no in vivo antitumor activity against the syngeneic B16 melanoma or MCA-203 sarcoma.(ABSTRACT TRUNCATED AT 250 WORDS)

Bryostatin 1-activated T cells can traffic and mediate tumor regression.

Adoptive immunotherapy in humans may be limited by the lack of autologous tumor cells to activate and expand tumor-specific T cells. Pharmacologic manipulation of protein kinase C (PKC) and intracellular calcium may substitute for tumor antigen and stimulate T cells for adoptive immunotherapy. In the present study, we evaluated the ability of the PKC activator Bryostatin 1 (B) plus the calcium ionophore ionomycin (I) to activate lymphocytes obtained from popliteal lymph nodes (DLN) draining an MCA-105 footpad tumor. The adoptive transfer of B/I-stimulated DLN cells eradicated MCA-105 pulmonary metastases. These lymphocytes do not require concomitant IL-2 administration to mediate regression of lung metastases. Three days after intrasplenic injection of tumor cells and splenectomy, mice were given iv injections of B/I-stimulated DLN cells. Adoptive immunotherapy with these cells induced regression of established liver metastases. In an intradermal tumor model, the adoptive transfer of B/I-stimulated MCA-105 DLN cells cured mice of MCA-105 intradermal (id) tumors, but did not induce regression of MCA-206 tumors. Mice cured of MCA-105 id tumors were protected against MCA-105, but not MCA-203, tumor challenge in the footpad 7 weeks after adoptive immunotherapy.

Effect of a combined exposure to cytosine arabinoside, bryostatin 1, and recombinant granulocyte-macrophage colony-stimulating factor on the clonogenic growth in vitro of normal and leukemic human hematopoietic progenitor cells.

We have examined the effect of a combined 24 h exposure to cytosine arabinoside (ara-C) and the protein kinase C activator bryostatin 1, either alone or in conjunction with recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), on the clonogenic growth of 14 primary samples from acute myelogenous leukemia (AML) patients, as well as normal human committed and early hematopoietic progenitors. Incubation of blasts with 1 microM ara-C and 12.5 nM bryostatin 1(+/- 1.25 ng/ml rGM-CSF) resulted in a heterogeneous pattern of inhibitory effects toward primary leukemic colonies, ranging from 32-98%, and subadditive to synergistic drug interactions. However, exposure of blasts to ara-C and bryostatin 1, either with or without rGM-CSF, eliminated leukemic cell self-renewal in 80-93% of samples, and very substantially reduced growth in the remainder. Exposure of normal human bone marrow mononuclear cells to identical concentrations of ara-C and byostatin 1 permitted the survival of 23% of committed myeloid progenitors (granulocyte-macrophage colony-forming units), and greater than 50% when rGM-CSF was included. Finally, exposure of bone marrow populations highly enriched for progenitor cells (CD34+, DR-, CD71-) to ara-C and bryostatin 1 +/- rGM-CSF for 24 h led to minimal reductions (e.g. 10-15%) in the survival of early hematopoietic progenitors (high proliferative potential colony-forming cells). Together, these findings indicate that combined exposure in vitro to ara-C and bryostatin 1, both with and without rGM-CSF, effectively inhibits the growth of leukemic cells with self-renewal capacity, while sparing a significant fraction of normal committed and primitive hematopoietic progenitors.

Corticosteroids inhibit the delivery of short-term activational pulses of phorbol ester and calcium ionophore to human peripheral T cells.

Although there is evidence that corticosteroids inhibit receptor-ligand-induced phospholipid hydrolysis, the immunosuppressive effects of these agents downstream of protein kinase C (PK-C) activation and cytosolic Ca2+ mobilization is unclear. Previous studies indicated that T cell proliferative activation could be achieved with simultaneous short-term (e.g., 15-120 min) exposure to agents activating PK-C and elevating cytosolic Ca2+. In the studies reported here, similar procedures were utilized for determining whether corticosteroids alter T cell activation signals downstream of second messenger events. Dexamethasone interfered with T cell activation induced by short-term exposure to phorbol 12,13-dibutyrate (PDBu) and the calcium ionophore, ionomycin. The inhibitory effect was evident with as little as 15 min of exposure to dexamethasone and T cell activating agents, making mechanisms involving de novo protein synthesis unlikely. Dexamethasone's effects in this system were blocked by the steroid receptor antagonist RU-486, indicating that the inhibition was mediated through the glucocorticoid receptor. The inclusion of recombinant interleukin-2 (IL-2) only partially overcame the dexamethasone inhibitory effect. Long-term (i.e., 48 hr) direct stimulation of PK-C with either PDBu or the non-tumor-promoting PK-C activator, bryostatin 1, also substantially overcame dexamethasone's effects, resulting in a recovery of IL-2 production and significant restoration of the T-cell proliferative response. These observations suggest that treatment with a PK-C-activating agent such as bryostatin 1 could reduce glucocorticosteroid-induced immunosuppression.

Activation and growth of murine tumor-specific T-cells which have in vivo activity with bryostatin 1.

We examined the ability of bryostatin 1 (Bryo), a novel protein kinase C activator, plus ionomycin (Io), a calcium ionophore, to activate T-cells with specific antitumor activity. Lymphocytes from the draining lymph nodes (DLN) of MCA-105 tumor-bearing host mice were stimulated with Bryo/Io, either fresh or after in vitro stimulation with autologous tumor, and then were incubated in interleukin-2 at 20 units/ml. Lymphocytes sensitized with tumor cells in vitro and then stimulated with Bryo/Io exhibited significant expansion (12-fold) after a total of 3 weeks in culture and moderate cytolytic activity (40% at an effector:tumor cell ratio of (80:1) and were exclusively CD8+ T-cells. DLN cells activated immediately with Bryo/Io, without tumor antigen sensitization in vitro, displayed marked growth (130-fold expansion) over 3 weeks in culture, had weak cytolytic activity (8% at an effector:tumor ratio of 80:1), and were a mixed population of CD8+ and CD4+ cells. Despite the differences in phenotypes and in cytotoxicity, both groups of DLN cells were highly effective in vivo against MCA-105 pulmonary metastases. Bryo/Io-activated DLN cells from MCA-105 tumor-bearing hosts had no therapeutic efficacy against B16 melanoma or MCA-203 sarcoma metastases. Lymph node cells from normal mice and non-draining lymph node cells from tumor-bearing hosts could be expanded with Bryo/Io to a degree similar to that of DLN cells but had no antitumor activity. Phenotypic analyses and in vitro and in vivo depletion studies demonstrate that CD8+ cells mediated tumor regression.

Effect of bryostatin 1 on the in vitro radioprotective capacity of recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) toward committed human myeloid progenitor cells (CFU-GM).

We have examined the effect of the macrocyclic lactone protein kinase C (PK-C) activator bryostatin 1 on the in vitro radioprotective capacity of recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF) toward normal committed myeloid progenitor cells (day-14 granulocyte-macrophage colony-forming units [CFU-GM]). Preincubation of T-cell- and adherent cell-depleted bone marrow mononuclear cells with 12.5 nM bryostatin 1 and either 1.25 or 50 ng/ml rGM-CSF for 24 h resulted in an 18%-30% survival at 4-5 Gy, whereas cells exposed to rGM-CSF alone gave rise to no detectable colonies at radiation doses greater than 2.5 Gy. Coadministration of bryostatin 1 also led to a threefold increase in Do values for both rGM-CSF concentrations. A similar enhancement of radioprotective effects was observed with the tumor-promoting phorbol ester phorbol dibutyrate. Exposure of cells to both bryostatin 1 and rGM-CSF immediately following irradiation also resulted in enhanced progenitor cell survival when compared to rGM-CSF alone, but radioprotective effects were less than those observed when cells were preincubated with these factors. Cells preconditioned with bryostatin 1 and rGM-CSF prior to exposure to 2 or 4 Gy gave rise to significantly more colonies when radiation was administered as a 4-h divided dose, suggesting that bryostatin 1 may act by potentiating rGM-CSF-induced repair of sublethal radiation damage. Finally, pre-exposure of enriched progenitor cells (CD34+) to bryostatin 1 and rGM-CSF resulted in radioprotective effects that were less than those observed for partially purified populations with respect to the total population of surviving myeloid colonies. However, CD34+ cells preincubated with bryostatin 1 and rGM-CSF prior to irradiation exhibited a significant increase in both the percentage and absolute number of neutrophilic and macrophage colonies, and a reduction in eosinophilic colonies, compared to cells exposed to rGM-CSF alone. These studies suggest that bryostatin 1 (and possibly other PK-C activators) potentiates the in vitro radioprotective effects of rGM-CSF and may also regulate the lineage specificity of this response.

Angiofollicular lymph node hyperplasia (Castleman's disease) in an adolescent female. Clinical and immunologic findings.

Angiofollicular lymph node hyperplasia is a heterogeneous disorder of unclear etiology and has a wide spectrum of systemic symptoms. This report describes a case of this disorder in a 15-year-old girl and examines the response of the primary mass, systemic symptoms, and alterations of selected immune parameters at diagnosis, as a result of steroid therapy and radiation therapy (RT). The patient had a 1-year history of growth failure, delayed puberty, and refractory iron deficiency anemia. Computed tomography scan showed a posterior mediastinal mass. Biopsy revealed angiofollicular lymph node hyperplasia of mixed hyaline-vascular and plasma cell type histologic type. Immunoperoxidase studies showed polyclonal B-cells, predominance of T-helper cells (CD4) over cytotoxic/suppressor T-cells (CD8), and the presence of natural killer (NK) cells. Southern blot analysis demonstrated germ line gene configuration for the T-cell antigen receptor and Ig heavy chain. The patient clinically improved with RT after failing to respond to steroids. Immunophenotyping of peripheral blood lymphocytes before therapy revealed a CD4:CD8 ratio of 0.8 with decreased numbers of circulating T-cells; this increased to 1.4 after steroid therapy. The patient's T-lymphocytes had no proliferative response to phytohemagglutinin (PHA) or concanavalin A (Con A) before RT. After RT, a small but significant mitogenic response to these reagents was noticed. The proliferative response to recombinant interleukin-2 (rIL-2) remained similar to that of control lymphocytes. Induction of second messenger signals by activation of protein kinase C (PKC) and elevation of free cytosolic calcium through the use of the phorbol ester, phorbol 12, 13-dibutyrate (PDBu), and ionomycin (Io) resulted in a strong proliferative response at diagnosis and after RT. In vitro cytotoxicity assays revealed diminished NK activity before and after therapy. Lymphokine-activated killer (LAK) activity remained comparable with that of control cells and was not affected by therapy. Before RT patient lymphocytes maintained cytotoxic capabilities after coincubation with rIL-2 and PDBu plus Io, whereas coincubation with these reagents abrogated cytotoxic function of normal cells. This case demonstrates a clinical response to RT as well as improvement in immune parameters. Intact signal transduction mechanisms through PKC activation and elevation of cytosolic calcium were also demonstrated in the circulating lymphocytes.

In vitro effects of bryostatin 1 on the metabolism and cytotoxicity of 1-beta-D-arabinofuranosylcytosine in human leukemia cells.

Bryostatin 1 is a macrocyclic lactone protein kinase C (PK-C) activator which has demonstrated promising antileukemic activity in preclinical studies. We have examined the effect of this agent on the metabolism and cytotoxicity of 1-beta-D-arabinofuranosylcytosine (ara-C) in both log phase and high-density human promyelocytic leukemia cells (HL-60). Exposure of low-density cells to 12.5 nM bryostatin 1 for 24 hr prior to a 4-hr incubation with 1 or 10 microM ara-C resulted in nearly a 2-fold increase in ara-CTP formation. When cells were maintained under high-cell density conditions (e.g. 5 x 10(6) cells/mL) for 24 hr prior to ara-C exposure, a 90% reduction in ara-CTP formation and ara-C DNA incorporation was observed. However, coincubation of high-density cells with bryostatin 1 for 24 hr increased ara-CTP formation 6- to 8-fold, yielding levels essentially equivalent to those achieved in low-density cells. Smaller (but still significant) increases in ara-C DNA incorporation were also noted. Enhancement of ara-CTP formation by bryostatin 1 occurred over a broad ara-C concentration range (0.1 to 100 microM), involved a temperature-dependent process, could not be mimicked by addition of hematopoietic growth factors, and was not related to neutralization of toxic or inhibitory substances in high-density medium. Exposure of cells to bryostatin 1 did not lead to morphologic or functional evidence of HL-60 cell maturation or an increase in cell viability, but did produce a decline in cellular proliferative activity as determined by thymidine and bromodeoxyuridine incorporation and cytofluorometric analysis. Bryostatin 1 did not exert its effects in high-density cells by inhibiting ara-C deamination or by interfering with ara-CTP dephosphorylation, but instead appeared to act by enhancing ara-C phosphorylation. Although cell-free extracts obtained from high-density cells exposed to bryostatin 1 exhibited levels of deoxycytidine kinase activity compared to controls, treated cells did display a significant decline in intracellular dCTP levels (e.g. 0.7 vs 1.3 pmol/10(6)), and nearly a 2-fold increase in ATP and UTP concentrations. Ara-CTP formation was also increased substantially by other PK-C activators including phorbol dibutyrate and mezerein (10-100 nM); this process was inhibited more than 70% by the PK-C inhibitor H-7 (50 microM), but not by the PK-C inhibitors staurosporine, tamoxifen, and HA1004. Finally, coadministration of ara-C and bryostatin 1 resulted in greater than expected inhibitory effects toward HL-60 cell clonogenic growth.(ABSTRACT TRUNCATED AT 400 WORDS)

A comparison of serum-free media for the support of in vitro mitogen-induced blastogenic expansion of cytolytic lymphocytes.

The high cost and potential dangers of including human AB serum in incubation media used to expand lymphocyte populations in vitro for adoptive immunotherapy have stimulated efforts to develop defined media which can support both the expansion and induction of lymphocytes with tumor cytolytic activity in the absence of serum. Lymphocyte proliferation following exposure to either PHA or the combination of phorbol 12,13-dibutyrate (PDBu) and the calcium ionophore, ionomycin, was evaluated. Although the media tested, X-Vivo 10, HB-104, AIM V, and HL-1, supported the generation of comparable levels of LAK activity after 3-5 days incubation with 10(3) U human recombinant interleukin-2 (rIL-2)/ml, there were striking differences in the ability of each medium to support mitogenically stimulated lymphocytes in the absence of serum, with cells in AIM V and X-Vivo 10 showing the highest levels of DNA synthesis. In long-term cultures (17 days) of blood MNC stimulated by PDBu and ionomycin, X-Vivo 10 and HB-104 yielded the greatest numbers of cells. The addition of 2% AB serum greatly enhanced the ability of each medium to support cell proliferation to equivalent maximum levels. The results indicate that while all four serum-free media were suitable for lymphocyte culture and support the development of LAK activity, they differ in their capacity to support expansion of lymphocyte populations in response to polyclonal mitogenic activation. This latter characteristic should be considered before choosing a particular serum-free formulation as its constituents may affect mechanistic interpretations regarding signal transduction events.

Effect of the protein kinase C activating agent bryostatin 1 on the clonogenic response of leukemic blast progenitors to recombinant granulocyte-macrophage colony-stimulating factor.

Bryostatin 1 is a macrocyclic lactone activator of protein kinase C which has displayed promising antileukemic potential in pre-clinical studies. We have assessed the effect of bryostatin 1 on the in vitro clonogenic response of leukemic myeloblasts obtained from 12 patients with acute non-lymphocytic leukemia to recombinant granulocyte-macrophage colony-stimulating factor (rGM-CSF), and have compared these responses to those of normal human hematopoietic progenitors. Although leukemic blast progenitors responded in a heterogenous manner to bryostatin 1 as a single agent, co-administration of 12.5 or 100 nM bryostatin 1 in conjunction with 1.25 ng/ml rGM-CSF resulted in a significant reduction in colony formation (compared to rGM-CSF alone) in 8/12 specimens, and sub-additive stimulatory effects in all samples. In addition, the exposure of cells to 12.5 nM bryostatin 1, either alone or in conjunction with 1.25 ng/ml rGM-CSF, substantially reduced or eliminated leukemic cell self-renewal capacity in all samples assayed. In contrast to the effects observed in leukemic cells, exposure of adherent and T-cell depleted normal bone marrow mononuclear cells to equivalent concentrations of bryostatin 1 and rGM-CSF consistently produced supra-additive effects on the growth of normal committed myeloid progenitors (day 14 CFU-GM). When normal marrow cells were further enriched for progenitors (MY-10+), concentrations of bryostatin 1 that were unable to support growth when administered alone significantly potentiated the number of GM colonies formed in response to rGM-CSF. These studies suggest that bryostatin 1 may modulate the in vitro response of certain normal and leukemic progenitor cells to rGM-CSF, and that the nature of this response differs between the two cell types. They also indicate that bryostatin 1 may be particularly effective in limiting the self-renewal capacity of leukemic myeloblasts, an in vitro characteristic with potentially important in vivo significance.

Activation and in vitro expansion of tumor-reactive T lymphocytes from lymph nodes draining human primary breast cancers.

The feasibility of in vitro activation of lymphocytes from the draining lymph nodes (DLN) of breast cancer patients was examined. Lymphocytes isolated from 48 DLN from 12 patients were examined for their proliferative responses to rIL-2, autologous tumor cells, or rIL-2 plus tumor cells. Three general patterns of cellular responses were observed. Cells from some DLN (17%) were unresponsive to any stimuli. Lymphocytes from 52% of the DLN responded moderately to rIL-2 alone. The combination of rIL-2 and tumor antigen had a synergistic effect on the proliferation of cells from 31% of the DLN assayed. Phorbol dibutyrate and ionomycin plus rIL-2 stimulated expansion of DLN lymphocytes by up to 850-fold after 35 days. These expanded cell populations, as well as those stimulated with antigen plus rIL-2, were predominantly CD3+ and CD16- cells, varying in proportions of CD4+ and CD8+ subsets. Both populations were cytotoxic against autologous tumor, MCF-7, and K562 target cells.

Effect of pharmacologic manipulation of protein kinase C by phorbol dibutyrate and bryostatin 1 on the clonogenic response of human granulocyte-macrophage progenitors to recombinant GM-CSF.

The effect of pharmacologic manipulation of protein kinase C (PK-C) activity on the response of committed human myeloid progenitor cells (CFU-GM) to recombinant human granulocyte-macrophage colony stimulating factor (rGM-CSF) was assessed. Coadministration of the PK-C activating agents, phorbol dibutyrate (PDBu) or bryostatin 1, with rGM-CSF resulted in a dose-dependent and, under some conditions, highly synergistic increase in the number of CFU-GM. With optimal combinations, colony formation far exceeded that which could be obtained with high concentrations of rGM-CSF alone. High concentrations of PDBu (e.g. greater than or equal to 50 nM), but not bryostatin 1, completely inhibited the CFU-GM response. These inhibitory effects could be reversed by bryostatin 1, but not by high concentrations of rGM-CSF. Bryostatin 1 also potentiated colony formation in response to rGM-CSF, and blocked the inhibitory effects of high concentrations of PDBu in bone marrow cells highly enriched for progenitors bearing the MY-10 antigen. The increase in CFU-GM induced by PDBu or bryostatin 1 was associated with little change in the morphologic type of colony observed. Continuous exposure of cells to the calcium ionophore, ionomycin (500 nM), reduced the number of granulocyte-macrophage colonies, but produced little change in the concentration-response of rGM-CSF and PK-C activating agents. Finally, the PK-C inhibitors H-7 and tamoxifen, when administered at concentrations exhibiting minimal inhibitory effects in the presence of rGM-CSF alone, led to no change or small increases in the numbers of colonies formed in response to rGM-CSF and bryostatin-1, and a substantial increase in the number of colonies formed in the presence of rGM-CSF and PDBu. These results suggest that PK-C activation may play a complex role in regulating the response of normal myeloid progenitors to growth factors such as rGM-CSF. They also raise the possibility that under some circumstances the phorbol ester PDBu may trigger events that inhibit the growth of myeloid progenitors, and that this process may be blocked by bryostatin 1.

Effect of tetrahydrouridine and deoxytetrahydrouridine on the interaction between 2'-deoxycytidine and 1-beta-D-arabinofuranosylcytosine in human leukemia cells.

The interaction between 2'-deoxycytidine (dCyd) and 1-beta-D-arabinofuranosylcytosine (ara-C), administered at pharmacologically achievable concentrations, was examined in four continuously cultured human leukemia cell lines, HL-60, KG-1, K-562, and CCRF-CEM. In three of the cell lines (HL-60, K-562, and CCRF-CEM), co-administration of 20 or 50 microM dCyd with 10 microM ara-C reduced ara-CTP formation by at least 90% and incorporation of ara-C into DNA by at least 80%. In contrast, KG-1 cells exhibited substantially smaller reductions in both ara-CTP formation and incorporation of ara-C into DNA under identical conditions. KG-1 cells were distinguished by the highest activity of the enzyme cytidine deaminase of the four lines assayed, and exhibited the smallest increments in the intracellular accumulation of both dCyd and deoxycytidine triphosphate (dCTP) in response to exogenous dCyd. Co-administration of 1 mM tetrahydrouridine (THU) or 0.5 mM deoxy-tetrahydrouridine (dTHU) had little effect on the ability of dCyd to antagonize ara-C metabolism in HL-60, KG-1 and K-562 cells. In contrast, these deaminase inhibitors substantially increased the intracellular accumulation of dCTP as well as the ability of dCyd to antagonize ara-CTP formation and incorporation of ara-C into DNA in KG-1 cells. THU and dTHU also permitted dCyd to antagonize ara-C growth inhibitory effects in KG-1 cells to the extent observed in the other leukemic cell lines. These studies suggest that the intracellular deamination of exogenous deoxycytidine may influence the degree to which this nucleoside antagonizes ara-C metabolism and toxicity in some leukemic cells. They also raise the possibility that deaminase inhibitors may be employed to modulate, and perhaps to improve, the therapeutic selectivity of pharmacologically relevant concentrations of ara-C and dCyd in the treatment of acute leukemia in man.