c-myc gene-induced alterations in protein kinase C expression: a possible mechanism facilitating myc-ras gene complementation.

The mechanism(s) by which the c-myc nuclear protein and the membrane-associated ras protein interact to mediate phenotypic changes is unknown. We now find that c-mcy gene expression is associated with alterations in the principal signal transduction pathway through which the ras protein is thought to function. We studied the transcript and protein expression of protein kinase C (PKC) isoforms in a culture line of human small cell lung cancer cells (NCI H209) in which expression of inserted c-myc and Ha-ras genes together, but not alone, causes a transition to a large cell phenotype. In control H209 cells, at the transcript and cell membrane protein levels, PKC-alpha is the dominant PKC species. In this cell line, the expression of an exogenous c-myc gene, but not of a viral Ha-ras gene, causes a 5- to 10-fold increase in the PKC-beta isoform transcript and protein. The insertion of ras into the exogenous myc-expressing 209 cells, in addition to causing phenotypic transition, results in the translocation of the PKC-beta protein from the cytosol to the membrane fraction and a decrease in membrane-associated PKC-alpha. Concomitant with these changes, the increased PKC isoform transcript levels induced by myc alone are completely reversed. These observations suggest that a complex set of PKC transcript and protein alterations, most prominently involving an increased PKC-beta protein level in the cell membrane, a decrease in PKC-alpha protein, and a decrease in all PKC isoform transcripts, may represent a fundamental event(s) for c-myc collaboration with Ha-ras to alter cell phenotype.

Differential effects of bryostatin 1 and phorbol ester on human breast cancer cell lines.

The effects of the protein kinase C (PKC) activators, phorbol ester 12-O-tetradecanoyl-13-phorbol acetate (TPA) and the marine natural product, bryostatin 1, on the growth and morphology of human breast cancer cell lines were examined. TPA (1 to 100 nM) inhibited growth of four of six cell lines by up to 75% in 5-day cultures. Bryostatin 1 inhibited growth of only MCF-7 cells and only at a high dose (100 nM). However, bryostatin 1 completely antagonized the growth inhibition and morphological changes induced by TPA in MCF-7 cells. The divergent effects of these two agents are associated with differing effects on PKC activity and isoform expression in MCF-7 cells. TPA induced rapid translocation of the PKC-alpha isozyme and PKC activity to the membrane fraction of MCF-7 cells. In contrast, bryostatin 1 treatment resulted in the loss of the PKC-alpha isozyme and PKC activity from both cytosolic and membrane compartments within 10 min of treatment. In coincubation assays the bryostatin 1 effect was dominant over that of TPA. Similar effects on PKC-alpha isozyme and PKC activity were seen in a second cell line whose growth was inhibited by TPA but not by bryostatin 1, MDA-MB-468. In contrast, in the T47D cell line, where TPA was not growth inhibitory, TPA failed to induce translocation of PKC-alpha to the cell membrane. Bryostatin, however, still caused loss of PKC-alpha isozyme and PKC activity from cytosolic and membrane fractions. Thus, differential actions of bryostatin 1 and TPA on PKC activity and alpha-isoform level in the membrane-associated fraction of MCF-7 and MDA-MB-468 cells may account for the divergent effects of these two agents on cell growth and morphology. These results suggest that the PKC-alpha isoform may specifically play a role in inhibiting growth of human breast cancer cells.

Bryostatin induces changes in protein kinase C location and activity without altering c-myc gene expression in human promyelocytic leukemia cells (HL-60).

When human promyelocytic leukemia cells (HL-60) are induced by phorbol esters to differentiate to macrophages, the process is accompanied by immediate activation of protein kinase C (PK-C) in the cytoplasm and later changes in DNA and RNA synthesis. Although these events are temporarily related, it remains unclear how activation of this protein kinase leads to changes in nuclear transcription. In this study, we find that bryostatin, a macrocyclic lactone which does not induce differentiation of HL-60 cells but activates PK-C, mimics the effects of phorbol esters on protein phosphorylation and PK-C location. Treatment of HL-60 cells with bryostatin stimulates phosphorylation of the surface transferrin receptor and in the cytoplasm of five proteins having the molecular weights of 17-43 kDa over the same time course as that stimulated by phorbol esters. Similarly, prolonged treatment with bryostatin, like that with phorbol esters, causes the loss of all cellular PK-C activity. Unlike the phosphorylation studies, bryostatin treatment, over a 1-100 nM concentration range and for varying lengths of time, did not affect HL-60 c-myc RNA levels, while phorbol ester treatment rapidly decreased c-myc RNA levels. These data suggest that neither the activation of PK-C and the phosphorylation of specific substrates nor the loss of total cellular PK-C activity from HL-60 cells is sufficient to induce marked decreases in c-myc levels and differentiation of HL-60 cells.

High-dose cytotoxic therapy and bone marrow transplantation for relapsed Hodgkin's disease.

Patients with Hodgkin's disease who have failed two or more chemotherapy regimens or who have relapsed after an initial chemotherapy-induced remission of less than 12 months are seldom cured with conventional salvage therapies. We studied the effect of high-dose cytoreductive therapy followed by bone marrow transplantation in 50 such patients with relapsed Hodgkin's disease. Twenty-one patients with histocompatibility locus antigen (HLA)-matched donors had allogeneic marrow transplants, one patient received marrow from an identical twin, and 28 patients without a matched donor received autologous grafts purged with 4-hydroperoxycyclophosphamide. Busulfan plus cyclophosphamide was the preparative regimen for the 25 patients who had received extensive prior irradiation, and the other 25 patients received cyclophosphamide plus total body irradiation. The overall actuarial probability of event-free survival at 3 years was 30%, with a median follow-up of 26 months. The event-free survival following transplantation was influenced by the number of chemotherapy failures and the patient's response to conventional salvage therapy prior to transplant. The 16 patients who were transplanted at first relapse, while still responsive to standard therapy, had a 64% actuarial probability of event-free survival at 3 years. Age, presence of extranodal disease, preparative regimen, and type of graft (autologous v allogeneic) were not significant prognostic factors. The majority of transplant-related deaths were from interstitial pneumonitis; inadequate pulmonary function, multiple prior chemotherapy regimens, and prior chest irradiation all appeared to increase the transplant-related mortality. These results suggest a role for marrow transplantation in a subset of patients with relapsed Hodgkin's disease who are unlikely to be otherwise cured but are still responsive to conventional-dose cytoreductive therapy.

Phosphorylation of Bcl2 and regulation of apoptosis.

Members of the Bcl2 family of proteins are important regulators of programmed cell death pathways with individual members that can suppress (eg Bcl2, Bcl-XL) or promote (eg Bax, Bad) apoptosis. While the mechanism(s) of Bcl2's anti-apoptotic function is not yet clear, introduction of Bcl2 into most eukaryotic cell types will protect the recipient cell from a wide variety of stress applications that lead to cell death. There are, however, physiologic situations in which Bcl2 expression apparently fails to protect cells from apoptosis (eg negative selection of thymocytes). Further, Bcl2 expression in patient tumor samples does not consistently correlate with a worse outcome or resistance to anticancer therapies. For example, patient response and survival following chemotherapy is independent of Bcl2 expression at least for pediatric patients with ALL. These findings indicate that simple expression of Bcl2 may not be enough to functionally protect cells from apoptosis. The finding that Bcl2 is post-translationally modified by phosphorylation suggests another level of regulation of function. Recent studies have shown that agonist-activated phosphorylation of Bcl2 at serine 70 (single site phosphorylation), a site within the flexible loop domain (FLD), is required for Bcl2's full and potent anti-apoptotic function, at least in murine IL-3-dependent myeloid cell lines. Several protein kinases have now been demonstrated to be physiologic Bcl2 kinases indicating the importance of this post-translational modification. Since Bcl2 phosphorylation has been found to be a dynamic process involving both a Bcl2 kinase(s) and phosphatase(s), a mechanism exists to rapidly and reversibly regulate Bcl2's activity and affect cell viability. In addition, multisite Bcl2 phosphorylation induced by anti-mitotic drugs like paclitaxel may inhibit Bcl2 indicating the potential wide range of functional consequences that this post-translational modification may have on function. While post-translational mechanisms other than phosphorylation may also regulate Bcl2's function (eg ubiquitination), this review will focus on the regulatory role for phosphorylation and discuss its potential clinical ramifications.

Serine/threonine phosphorylation in cytokine signal transduction.

Over the past decade, the involvement of tyrosine kinases in signal transduction pathways evoked by cytokines has been intensively investigated. Only relatively recently have the roles of serine/threonine kinases in cytokine-induced signal transduction and anti-apoptotic pathways been examined. Cytokine receptors without intrinsic kinase activity such as interleukin-3 (IL-3), granulocyte-macrophage colony-stimulating factor (GM-CSF) and the interferons were thought to transmit their regulatory signals primarily by the receptor-associated Jak family of tyrosine kinases. This family of tyrosine kinases activates STAT transcription factors, which subsequently transduced their signals into the nucleus to modulate gene expression. Cytokine receptors with intrinsic tyrosine kinase activity such as c-Kit were initially thought to transduce their signals independently of serine/threonine kinase cascades. Recently, both of these types of receptor signaling pathways have been shown to interact with serine/threonine kinase pathways as maximal activation of these tyrosine kinase regulated cascades involve serine/threonine phosphorylation modulated by, for example MAP kinases. A common intermediate pathway initiating from cytokine receptors is the Ras/Raf/MEK/ERK (MAPK) cascade, which can result in the phosphorylation and activation of additional downstream kinases and transcription factors such as p90Rsk, CREB, Elk and Egr-1. Serine/threonine phosphorylation is also involved in the regulation of the apoptosis-controlling Bcl-2 protein, as certain phosphorylation events induced by cytokines such as IL-3 are anti-apoptotic, whereas other phosphorylation events triggered by chemotherapeutic drugs such as Paclitaxel are associated with cell death. Serine/threonine phosphorylation is implicated in the etiology of certain human cancers as constitutive serine phosphorylation of STATs 1 and 3 is observed in chronic lymphocytic leukemia and can be inhibited by the chemotherapeutic drug fludarabine. Serine/threonine phosphorylation also plays a role in the etiology of immunodeficiencies. Activated STAT5 proteins are detected in reduced levels in lymphocytes recovered from HIV-infected individuals and immunocompromised mice. Serine/threonine phosphorylation may be an important target of certain chemotherapeutic drugs which recognize the activated proteins. This meeting report and mini-review will discuss the interactions of serine/threonine kinases with signal transduction and apoptotic molecules and how some of these pathways can be controlled by chemotherapeutic drugs. Leukemia (2000) 14, 9-21.

BAX and PKCalpha modulate the prognostic impact of BCL2 expression in acute myelogenous leukemia.

Previously, we demonstrated that the level of BCL2 expression is prognostic in acute myelogenous leukemia (AML). High levels of BCL2 correlate with an adverse outcome when associated with favorable and intermediate prognosis cytogenetics (FIPC), whereas low levels portend an adverse outcome when associated with unfavorable cytogenetics (UC). Because BCL2 function can be modulated by dimerization with family members, like BAX, or by phosphorylation by protein kinase C alpha (PKCalpha), we hypothesize that the relative expression of these proteins in primary leukemic cells might alter the prognostic impact of BCL2 expression. We therefore measured BAX and PKCalpha protein levels in peripheral blood mononuclear cell lysates from 165 newly diagnosed AML patients and correlated the expression of these proteins with BCL2 expression, patient survival, and remission induction success. Expression levels of BAX and PKCalpha were normalized against a control cell line, K562. BAX and PKCalpha expression levels were heterogeneous and did not correlate with the percentage of blasts in the sample (R2 = 0.01 and <0.01). The median expression of both was similar across FAB groups but the range was greater for M4. A similar distribution of expression was observed in all cytogenetic groups, except that patients with inversion 16 demonstrated lower levels of BAX. Individually, neither PKCalpha nor BAX expression was prognostic of response to induction therapy or survival. A similar outcome was obtained when patients were stratified by cytogenetics into FIPC and UC groups. However, the ratio of either BCL2:BAX (B2:BX) or PKCalpha*B2:BX (PK*B2:BX) was highly prognostic. Patients with FIPC and a lower ratio (less than median) of either B2:BX or PK*B2:BX had a significantly higher remission induction rate (88 versus 69%, P = 0.04) and longer survival (median: 141 versus 80.5 weeks, P = 0.007) compared with those with ratios more than median. For patients with UC, values of either B2:BX or PK*B2:BX below the median had an inferior response rate to induction therapy (35 versus 78%, P = 0.0006) and inferior survival outcomes (median survival: 11 versus 53 weeks, P = 0.00002). Interestingly, FIPC and UC patients with antiapoptotic ratios (defined as B2:BX or PK*B2:BX more than median) had identical response rates and survival outcomes. In multivariate analyses, the compound variables of cytogenetics and B2:BX, or PK*B2:BX were independent predictors of survival. These results suggest that expression levels of proteins that affect the functional status of BCL2 modify the prognostic impact of BCL2 and suggest that the role of apoptosis in different cases of AML varies independently in the different cytogenetic subgroups.

Familial association of giant cell arteritis. A case report and brief review.

The initial report of a father-daughter pair with giant cell arteritis (GCA) is made. To our knowledge, it represents only the 12th known familial aggregation of polymyalgia rheumatica (PMR) and/or GCA. This provides evidence for direct transmission of GCA during the two generations and helps to establish a genetic mode of inheritance for this disease. Clinically, the familial and nonfamilial forms of PMR and/or GCA are indistinguishable.

Regulation of CD34 expression in differentiating M1 cells.

CD34 is a cell surface glycoprotein expressed on hematopoietic stem and progenitor cells, but not on mature blood cells. In the present study we found that CD34 downregulation during hematopoiesis occured at the level of transcriptional initiation. Two transcription initiation sites (TISs) were identified in each of three different CD34+ cell lines; these TISs were located at 120 and 80 bp 5' of the translation start site, respectively. The promoter lacks TATA elements and, like other TATA-less promoters, the TISs conform to the consensus sequence for an INR (PyPyCAPyPyPyPy). An additional 3000 bp of upstream genomic DNA were sequenced and found to contain consensus sites for transcription factors, suggesting their potential role in gene regulation. Transient transfection assays using CD34 promoter-luciferase reporter constructs, containing sequences up to 3 kb upstream and inclusive of the TIS, indicate that this promoter drives transcription in hematopoietic CD34+ cells but not CD34+ nonhematopoietic cells. Both cell type specific expression and full promoter activity are maintained in constructs that contain as little as 454 bp upstream of the TISs. Optimal promoter activity requires the 5' untranslated region of exon 1, which contains a 51-bp element that has the potential to form an extensive secondary structure. In the plasmid DNA, however, this secondary structure was not detectable by P1 nuclease digestion. At least three proteins present in uninduced M1 nuclear extracts bind to this element. Two of the three proteins were identified as Sp 1 and Sp 3 based on supershift experiments. These data suggest that CD34 expression by hematopoietic stem and progenitor cells involves hematopoietic cell-specific factors that interact with regulatory elements within the first 230 bp of the promoter and that optimal expression requires a 60-bp segment of the 5' untranslated region.

CD34 expression by embryonic hematopoietic and endothelial cells does not require c-Myb.

CD34 is a cell-surface glycoprotein expressed in a developmental, stage-specific manner by bone marrow stem and progenitor cells. In this study we explored a possible role for c-Myb in CD34 regulation during developmental hematopoiesis. The results indicate that c-Myb can induce CD34 expression in hematopoietic and nonhematopoietic cells, and that murine CD34 promoter activity is enhanced in myeloid cells transgenic for c-Myb. To test whether c-Myb is necessary for CD34 expression during developmental hematopoiesis in vitro, c-Myb-null D3 embryonic stem (ES) cells were analyzed for their ability to develop CD34+ hematopoietic cells in vitro. CD34 promoter activity in transient transfections and CD34 upregulation during ES cell differentiation into embryoid bodies was identical in wild-type and c-Myb-null ES cells, indicating that c-Myb is not required for CD34 expression. CD34 protein is expressed on both hematopoietic and endothelial cells of the E8.5 blood islands during the development of c-Myb-null embryos, and expression is nearly identical in wild-type and c-Myb-null embryos. However, in E12.5 c-Myb-null embryos, the majority of identifiable CD34+ cells in the developing liver are endothelial rather than hematopoietic, which is consistent with the absence of colony-forming units in c-Myb-null embryos and developing ES cells. These data indicate that c-Myb is not required for CD34 expression in endothelial or primitive hematopoietic cells in the yolk sac, but is necessary for definitive hematopoiesis.

Regulation of Bcl2 phosphorylation and potential significance for leukemic cell chemoresistance.

Although considered tightly linked, the linkage effectors for proliferation and antiapoptotic signaling pathways are not clear. Phosphorylation of Bcl2 at serine 70 is required for suppression of apoptosis in interleukin 3 (IL-3)-dependent myeloid cells deprived of IL-3 or treated with antileukemic drugs and can result from agonist activation of mitochondrial protein kinase C alpha (PKCalpha). However, we have recently found that high concentrations of staurosporine up to 1 microM: can only partially inhibit IL-3-stimulated Bcl2 phosphorylation but completely block PKCalpha-mediated Bcl2 phosphorylation in vitro, indicating the existence of a non-PKC, staurosporine-resistant Bcl2 kinase (SRK). Although the RAF-1MEK-1-mitogen-activated protein kinase (MAPK) cascade is required for factor-dependent mitogenic signaling, a direct role in antiapoptosis signaling is not clear. In particular, the role of phosphorylation in the regulation of death substrates is not yet clear. Our findings indicate a potential role for the MEK/MAPK pathway in addition to PKC in antiapoptosis signaling, involving Bcl2 phosphorylation that features a role for extracellular signal-regulated kinase (ERK)1 and 2 as SRKs. These findings indicate a novel role for ERK1 and 2 as molecular links between proliferative and survival signaling and may, at least in part, explain the apparent paradox by which Bcl2 may suppress staurosporine-induced apoptosis. Although the effect of phosphorylation on Bcl2 function is not clear, effector molecules that regulate Bcl2 phosphorylation may have clinical significance in patients with acute myelogenous leukemia (AML) who express detectable levels of Bcl2. Preliminary findings suggest that expression of PKCalpha, ERK2, and Bax in leukemic blast cells from patients with AML, although individually not prognostic, appears to have potential clinical value in predicting chemoresistance and survival outcomes.

BMT for severe aplastic anemia using cyclosporine.

Between 1984 and 1991 24 patients with severe aplastic anemia (SAA) were transplanted with HLA identical sibling donor BM. The overall long-term survival was 79 +/- 8%. The average age was 21 years (range 4-53 years) and the median pre-transplant disease duration was 35 days (range 12-2998 days). Over one-half (15 of 24) of the patients had received > 10 units of blood product transfusions prior to BMT. The pre-transplant conditioning regimen consisted of 200 mg/kg cyclophosphamide (CY). Cyclosporine (CYA) was administered from 2 days prior to BMT and continued for 6-12 months. Two of the 24 patients failed to achieve primary engraftment (FTE). One of these patients had autologous recovery of BM function and is alive and well. Five of the 22 patients who engrafted failed to sustain engraftment (FTSE). Of these, three are alive and well following a second BMT or marrow boost. Only 1 of the 22 patients who engrafted had clinically significant (i.e. Stage II-IV) acute GVHD. No patient developed chronic GVHD. Our results indicate that BMT following a regimen consisting of CY with the continuous use of CYA in the post-transplant period is well tolerated and associated with excellent long-term survival. The high incidence of secondary graft instability (i.e. FTSE), however, suggests that future studies should focus on post-transplantation immunomodulation.

Activated protein kinase C directly phosphorylates the CD34 antigen in acute lymphoblastic leukemia cells.

The precursors of all blood cell lineages are contained within the 1-3% of bone marrow cells which express the CD34 antigen, and this population can reconstitute the hematopoietic system of lethally irradiated animals and humans. A potential regulatory role for the CD34 antigen in progenitor cell function and differentiation was indicated by our recent findings that the CD34 antigen can be phosphorylated in vivo to high stoichiometry in primitive CD34+ cell-lines by activated protein kinase C. To exclude the possibility that these effects were restricted to cell-lines, we have performed similar experiments on fresh cells from a patient with drug-resistant acute lymphoblastic leukemia. Similar to our previous findings, we found the CD34 antigen to be hyperphosphorylated in lymphoblasts labeled in the presence of active phorbols. The same peptides which were hyperphosphorylated in phorbol-stimulated cell-lines were also phosphorylated in phorbol-stimulated lymphoblasts. These data indicate that CD34 is a substrate molecule for PKC in fresh CD34+ lymphoblasts and underline the role of modulators of PKC activity in the biology of primitive leucocytes.

Treatment of refractory acute leukemia with timed sequential chemotherapy using topotecan followed by etoposide + mitoxantrone (T-EM) and correlation with topoisomerase II levels.

A phase I/II clinical study evaluated 17 patients with refractory/recurrent acute leukemia treated with 1.5 mg/m2/day topotecan on days 1-3 followed by etoposide (100 mg/m2/day)+mitoxantrone (10 mg/m2/day) on days 4, 5 and 9, 10. Timed sequential chemotherapy using the topoisomerase I-inhibitor topotecan before the topoisomerase II-inhibitors, etoposide+mitoxantrone (T-EM) treatment is proposed to induce topoisomerase II protein levels and potentiate the cytotoxic activity of the topoisomerase II-directed drugs. Fourteen patients had refractory and three had recurrent acute leukemia. The majority of patients were heavily pre-treated with greater than three re-induction chemotherapy regimens. Ten patients responded to T-EM treatment (59%). Four of seventeen (24%) had a complete remission and one had a partial remission. Four additional patients (24%) who scored complete leukemia clearance had no evidence of disease with complete white and red blood cell recovery but with platelet counts less than 100,000. The lack of platelet recovery in one patient having a partial response was scored as a partial leukemia clearance. The toxicity profile included major non-hematological toxicity including grade 3 mucositis (29%) and neutropenic fever (65%). Paired measurements of intracellular levels of topoisomerase II isoforms alpha and beta in leukemia blast cells (bone marrow) collected before (day 0) and after topotecan treatment (day 4) showed that a relative increase of topoisomerase IIalpha (Topo IIalpha) > or = 40% strongly correlated with response after T-EM treatment. Increased Topo IIalpha levels also corresponded to increased DNA fragmentation. Two patients who had an increase of Topo IIalpha of 20-25% had either a PR or PLC while patients with a < 10% increase showed no response to T-EM treatment. We conclude that timed sequential chemotherapy using topotecan followed by etoposide+mitoxantrone is an effective regimen for patients with refractory acute leukemia, and demonstrate Topo IIalpha protein level increases after topotecan treatment.

In vitro effects of hyperbaric oxygen on sickle cell morphology.

STUDY OBJECTIVE: To determine in vitro whether hyperbaric oxygen has any effect on the morphology of sickle cells. DESIGN: Prospective, in vitro, study, with each patient sample serving as its own control. SETTING: University medical center. PATIENTS: 10 children known to be homozygous for hemoglobin S. INTERVENTIONS: Blood samples were obtained from 10 children during routine visits to the University sickle cell clinic. Blood samples were exposed to room air to achieve maximal sickling. Each sample was divided into control and study aliquots, and the study portions placed in a research hyperbaric chamber with 100% oxygen at 3 atmospheres absolute pressure for 15 min. Then smears were prepared from all samples at regular intervals and examined by technicians in the sickle cell clinic who were blinded as to the details of this study. MEASUREMENTS: Percentages of normal cells, sickle cells and sickle forms were reported. Data were interpreted using t-tests. MAIN RESULTS: Hyperbaric oxygen appeared to have no effect on sickle cell morphology. Percentages of each cell type were unaffected by hyperbaric oxygen exposure. CONCLUSIONS: Hyperbaric oxygen appears to have no effect on the morphology of sickle cells in vitro. Other mechanisms may account for the beneficial clinical effects of hyperbaric oxygen in sickle cell crisis, although in vivo studies are warranted.

Respiratory monitoring.

Scientific advances over the last 20 years have enabled us to monitor the respiratory status of our patients beyond the age-old methods taught in physical diagnosis. It is now possible to measure almost every parameter of ventilation, oxygen transport, and CO2 transport, thus enhancing patient safety and patient care.