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Biomedical subjects

M Andreeff

Publications and source records attributed to M Andreeff.

At least 91 records · Page 5Linked to original sources
PubMed1998

Differential p53 phosphorylation and activation of apoptosis-promoting genes Bax and Fas/APO-1 by irradiation and ara-C treatment.

In this study, we examined the effects of radiation and ara-C on induction of apoptosis and on the apoptosis-promoting genes p53, Bax and Fas/APO-1, in BV173 human leukemia cells, which harbor the wild-type p53 gene. It has been reported that p53 upregulates Fas/APO-1 and Bax expression. Both irradiation and ara-C treatment resulted in apoptosis and induction of p53 proteins within hours. The Bax gene was activated in irradiated and ara-C-treated BV173 cells, but Fas/APO-1 was induced only in irradiated BV173 cells. Radiation and ara-C treatment did not induce Bax or Fas/APO-1 protein expression in p53-null HL60 cells. Radiation weakly induced Fas/APO-1 expression in KBM-7 cells, which harbor a partially defective p53 gene. Both HL60 and KBM-7 cells are more resistant to radiation- and ara-C-induced apoptosis than BV173 cells. These results suggest that functional p53 is necessary for the activation of Bax and Fas/APO-1 expression. However, elevated p53 protein is not sufficient to activate Fas/APO-1 gene expression in ara-C-treated cells. Using two-dimensional gel electrophoresis, we found that the p53 proteins in irradiated and ara-C-treated BV173 cells have different isoelectric points; they converged to a single isoelectric point after in vitro treatment with phosphatase. These results suggest that different genotoxic treatments cause different phosphorylations of p53, which may account for the different levels of activation of Fas/APO-1 expression.

Apoptosis↗
PubMed1998

Multilineage involvement of Philadelphia chromosome positive acute lymphoblastic leukemia.

Acute lymphocytic leukemia (ALL) is considered a clonal disease restricted to the lymphoid compartment. The Philadelphia chromosome (Ph) is found in a subset of ALL with poor prognosis. Here we present the largest series of Ph+ ALL analyzed for involvement of the myeloid compartment. For the first time at a single cell level the presence of Ph in lineages other than lymphoid is demonstrated. Granulocytes from nine patients diagnosed with BCR-ABL + ALL (eight Ph+, one Ph-) were purified using two layer density gradient separation. They were further identified by the morphology of DAPI-stained nuclei and studied for the presence of the Ph by fluorescence in situ hybridization (FISH) using a BCR-ABL dual-color probe. Ph was demonstrated in 30 to 93% of granulocytes in all patients. FISH identified major and minor BCR gene breakpoints (M-bcr and m-bcr). In one patient, with CD19+/34+/33-/2-/3-/7-/10- lymphoblasts, involvement of B cells (CD19+), T cells (CD3+), myeloid (CD13+), erythroid (glycophorin A+) cells was found by FISH following fluorescence-activated cell sorting (FACS). The diagnosis of ALL as opposed to lymphoblastic transformation of CML was established based on clinical and laboratory data including Western blot results demonstrating the presence of p190/m-bcr in five of the nine cases studied. Results suggest that Ph+ ALL originates from a pluripotent stem cell.

Adult↗
PubMed1998

Elevated levels of biologically active soluble CD40 ligand in the serum of patients with chronic lymphocytic leukaemia.

Chronic lymphocytic leukaemia (CLL) is an indolent lymphoproliferative disorder manifested by low growth fraction and prolonged survival of the malignant cells. The mechanisms that enable CLL cells to live longer and to resist apoptosis remain unclear. Because the malignant CLL cells express CD40 and Fas receptors, which can transduce cell-survival and cell-death signals, we examined the role of CD40 in the growth regulation of CLL cells and its interaction with Fas-mediated and fludarabine-induced apoptosis in vitro. Primary CLL cells underwent spontaneous apoptosis in culture, which was enhanced by exogenous human Fas ligand (FasL) or fludarabine. Exogenous CD40L rescued CLL cells from spontaneous apoptosis in a dose-dependent manner, and caused CLL cells to resist apoptosis induced by FasL or fludarabine. Patients' autologous plasma rescued CLL cells from spontaneous apoptosis, an effect that could be reversed with anti-CD40 ligand (CD40L) antibodies. The levels of soluble CD40 ligand in the sera of 51 CLL patients and 55 healthy donors were determined by enzyme-linked immunosorbent assay. The mean soluble CD40L level in normal donors was 0.29 ng/ml compared to a mean value of 0.80 ng/ml in CLL patients (P < 0.001). CD40L up-regulated bcl-X(L) mRNA but not bcl-2 in CLL cells within 3-6 h in culture. Our results demonstrated that serum of patients with CLL contained elevated levels of biologically active soluble CD40L, and that CD40L can prolong survival of CLL cells and mediate their resistance to FasL and fludarabine in vitro.

Adult↗
PubMed1998

Unbalanced expression of Fas and CD40 in mantle cell lymphoma.

B cells are characterized by the dual expression of CD40 and Fas receptors, which can mediate their survival and death, respectively. The balance between the dynamically opposing functions of these two receptors is important for B-cell selection, maturation and homeostasis. We found that mantle cell lymphoma (MCL) cells had a high level of CD40 and low or absent level of Fas, therefore favouring the CD40 cell survival pathway. Exogenous Fas ligand had no effect on MCL cells, whereas exogenous CD40 ligand enhanced their survival and rescued them from fludarabine-induced apoptosis. Our data raise the possibility that the prolonged survival of MCL cells in vivo may be explained by the unbalanced expression of Fas and CD40.

Apoptosis↗
PubMed1998

Coexpression of CD40 and CD40 ligand in B-cell lymphoma cells.

CD40 ligand (CD40L) is involved in the T-cell-dependent regulation of B-cell growth and survival and can rescue normal germinal centre B cells and several types of malignant B cells from apoptosis in vitro. We have previously reported that serum of patients with chronic lymphocytic leukaemia contained elevated levels of biologically active soluble CD40L (sCD40L). Whether an augmented CD40L pathway exists in patients with other types of B-cell lymphoid malignancies and the source of native sCD40L in these patients is currently unknown. Using a sensitive ELISA assay, soluble CD40L (sCD40L) was detected in the sera of both healthy individuals and patients with haematological malignancies; however, its level was significantly elevated only in patients with B-cell lymphomas (P<0.0001). Several types of malignant B cells coexpressed CD40 and CD40L proteins, and CD40L mRNA was detected in purified resting malignant B cells. The dual expression of CD40 and CD40L in B cells and the presence of native sCD40L in human serum suggest that a direct T-B-cell contact may not be required for CD40L delivery to B cells. This data raises the possibility that an autocrine cytokine loop involving CD40L may contribute to the growth regulation of benign and malignant B cells in vivo.

B-Lymphocytes↗
PubMed1998

CECA-cyclophosphamide, etoposide, carboplatin and cytosine arabinoside--a new salvage regimen for relapsed or refractory acute myelogenous leukemia.

In an effort to develop more effective therapy for patients with refractory or relapsed acute myelogenous leukemia (R-AML) we combined three drugs with proven activity in AML, that are not typically used in induction regimens, with cytosine arabinoside (ara-C). Twenty-five patients (3 primary refractory, 22 relapsed) were treated. Patients received 3 days of "CECA" therapy as follows: cyclophosphamide (CTX) 1 g/m2 i.v. over 2 hrs., etoposide (VP-16) 200 mg/m2 i.v. over 3 hr, carboplatin (CBP) 150 mg/m2 i.v. over 24 hours and ara-C 1 g/m2 over 2 hr. Peripheral circulating blasts cleared in 24 cases (96%), and marrow aplasia was achieved in 19 (76%). There were 3 complete remissions (CR), 1 patient died before day 14, 5 died aplastic 14 or more days from the start of therapy, 5 had primary resistant disease, and 10 had secondary resistance i.e., leukemia reappearing after developing aplasia and 1 was lost to follow up 6 weeks into therapy. Two of the patients achieving CR received allogeneic BMT in CR (at 18 and 22 weeks): one died of fungal infection on day 50 and the other, who had CNS involvement at relapse, is alive 24 months post transplant. Toxicity was tolerable: one patient each developed grade III diarrhea and mucositis, another had grade III cardiac toxicity, a fourth developed a grade IV bilirubin elevation. Single, 2, and 3 or more infectious episodes occurred in 10, 5 and 4 patients respectively. This regimen showed definite anti-leukemic activity: the 3 patients achieving CR were among 23 patients with a 1%, 10% or 20% expectation for second CR attainment. The CECA regimen should be investigated in better prognosis salvage groups.

Adolescent↗
PubMed1998

Rescue from apoptosis in early (CD34-selected) versus late (non-CD34-selected) human hematopoietic cells by very late antigen 4- and vascular cell adhesion molecule (VCAM) 1-dependent adhesion to bone marrow stromal cells.

Monoclonal antibodies to very late antigen 4 (VLA-4) recognize the alpha4beta1 integrin receptor. This monoclonal antibody blocks the adhesion between early hematopoietic progenitor cells (CD34-selected cells) and stromal cells when added to cultures of these cells. Addition of the VLA-4 monoclonal antibody to cultures of stromal cells and CD34-selected cells was shown to induce apoptosis of CD34-selected cells in these CD34-selected cell/stromal cell cocultures, as measured by the terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick end-labeling method. In contrast to these experiments with early hematopoietic progenitor cells (CD34+), the level of adhesion between more differentiated cells (unfractionated hematopoietic cells) and stromal cells was not significantly altered by addition of the anti-VLA-4 monoclonal antibody. Similarly, the level of apoptosis of unfractionated hematopoietic cells was not significantly increased by the addition of anti-VLA-4 monoclonal antibody to cultures of the latter cells with stromal cells. The binding of the unfractionated cells is less than that of the CD34-selected cells. Given that there is no difference between the alpha4beta1 integrin expression level of the early and late myeloid cells, there may be a difference in the functional state of the integrin between the early and late myeloid cells. We also show that CD34+-selected precursor cells proliferate at a higher rate when these cells are plated on recombinant vascular cell adhesion molecule 1 molecules. These data indicate that the alpha4beta1 integrin receptor (VLA-4) plays a central role in the apoptosis rescue function that results from the anchorage-dependent growth of the CD34-selected early hematopoietic cells on stromal cells. The data suggest that these apoptosis rescue pathways have less significance as the cells mature and become anchorage independent in their growth. These data should assist in the design of systems for the ex vivo proliferation and transduction of early hematopoietic cells for genetic therapy.

Antibodies, Monoclonal↗
PubMed1998

Ratio of bcl-xshort to bcl-xlong is different in good- and poor-prognosis subsets of acute myeloid leukemia.

BACKGROUND: Acute myeloid leukemia (AML) is a heterogeneous collection of leukemic disorders ranging from chemotherapy-sensitive subsets [inversion 16 and t(8;21)], which often can be cured with cytosine arabinoside alone, to the most resistant subsets, which can survive even supralethal levels of combination alkylator chemotherapy (cytogenetic subsets monosomy 5 and monosomy 7). MATERIALS AND METHODS: To analyze the expression of BCL-2 family genes, which are expressed in these subsets of AML, we used PCR sequence amplification reactions that are dependent on oligonucleotide primers representing the BH1 and BH2 homology domains to generate the unique regions between BH1 and BH2. These primers are conserved among all members of the BCL-2 gene family and are separated by a 150 nucleotide region sequence between the BH1 and BH2 domains. The PCR products unique to each BCL-2 family member were cloned directionally into sequencing vectors. The identity of the insert of each clone was determined by slotblots of the DNA amplified from individual colonies and by hybridization with radioactive probes specific to the bcl-2, bcl-x, or bax genes. RESULTS: We found that bcl-2 is the predominant member expressed in AML samples with a poor prognosis (-5, -7), whereas the transcripts of bcl-x are higher than those of bcl-2 in the AML samples with a good prognosis [inv16, t(8;21)]. No significant difference in bax expression was detected between AML subsets of good and bad prognosis. The ratio of bcl-xlong, which inhibits apoptosis, to bcl-xshort, which promotes apoptosis, was determined by amplification with a pair of primers specific to bcl-x followed by separation of the PCR product on agarose gels. Bcl-xlong and bcl-xshort appeared as bands of different molecular mass on a molecular weight gel and were visualized by ethidium bromide staining or Southern blot analysis with a bcl-x-specific probe. CONCLUSIONS: We found that the ratio of bcl-x long to bcl-x short was higher in the AML patients with a poor prognosis. These experiments showed that the levels of BCL-2 family members in the leukemia cells of good- and poor-prognosis subsets are different. In addition, novel members of the BCL-2 family were isolated from the cells of AML patients of either prognosis.

Acute Disease↗
PubMed1998

Low and maximally phosphorylated levels of the retinoblastoma protein confer poor prognosis in newly diagnosed acute myelogenous leukemia: a prospective study.

A prior retrospective study suggested that the level of retinoblastoma protein (RB) expression was prognostic for survival in acute myelogenous leukemia (AML). Individuals with no/low RB protein expression were considered to have loss of RB function, and those with maximally phosphorylated (maxphos) RB were also felt to have nonfunctional RB. To confirm this, we prospectively investigated whether the level of RB expression was prognostic in AML in a larger cohort of patients. RB level was measured by Western blot and immunohistochemical analysis on peripheral blood samples from 210 newly diagnosed AML patients. Patients were divided into three groups based on the level of RB protein expression (i.e., no or low, elevated, and maxphos) or into two groups on the basis of presumed RB function, altered function (AF-RB, low and maxphos RB), versus normal function (NF-RB, elevated RB). By combined results of Western blot and immunohistochemical analysis, 20%, 65%, and 15% of patients had low, elevated, and maxphos RB, respectively. Most patients with acute promyelocytic leukemia (APL) with a French-American-British classification of M3 were in the low RB group, likely reflecting a lower proliferative rate of promyelocytes. Analysis was performed with and without these APL patients. The median survival was significantly shorter for both patients with low RB expression (48 weeks, P = 0.05, including APL patients; 34 weeks, corrected P = 0.008, with APL patients excluded) and maxphos RB expression (51 weeks, P = 0.007) compared to those with elevated RB expression (122 weeks including and 98 weeks excluding APL patients). Differences were greatest among patients with nonfavorable prognosis cytogenetics (median survival, 34 weeks versus 85 weeks; corrected P = 0.001 for AF-RB versus NF-RB). Remission duration was also significantly shorter for non-APL patients with AF-RB versus NF-RB (median survival, 36 weeks versus not reached; corrected P = 0.02). In multivariate analyses, including cytogenetics, performance status, age, antecedent hematological disorder, and RB status, with and without APL patients included, no/low and maxphos-RB protein expression were independent predictors for poorer survival. This prospective study confirms that the level of expression of RB is a strong prognostic factor in AML, with an inferior survival experience being associated with no/low RB and maxphos RB expression. Therefore, therapeutic decisions based on the level of RB expression may be indicated, and protocols to incorporate this are currently under development.

Adolescent↗
PubMed1998

Humanized M195 monoclonal antibody conjugated to recombinant gelonin: an anti-CD33 immunotoxin with antileukemic activity.

The recently characterized immunotoxin HuM195-gelonin consists of a humanized anti-CD33 monoclonal antibody conjugated to the single-chain plant toxin gelonin. Binding of the immunotoxin to hematopoietic cells that express the CD33 differentiation antigen has been demonstrated and results in cytotoxicity due to ribosomal inactivation by gelonin. Blast cells from most patients with acute myelogenous leukemia express CD33, whereas normal stem cells necessary for maintenance of hematopoiesis do not. We asked whether an immunoconjugate using recombinant gelonin rather than plant gelonin is toxic to acute myelogenous leukemia (AML) cell lines and primary AML blasts obtained from patients and exposed to the immunotoxin in vitro. The CD33pos cell lines HL60, OCI/AML2, and OCI/ AML5 showed decreased proliferation when exposed to immunotoxin for 24-72 h. The CD33neg cell line OCI/AML3 was relatively resistant to HuM195, and all cell lines were resistant to equimolar concentrations of unconjugated antibody and gelonin. Primary blast cultures from seven patients with AML had CD33 detectable on 75.7-99.8% of cells by flow cytometry, and all showed dose-dependent decreases in clonogenic cell survival during 24-h incubation with the immunotoxin. Cells selected for low CD33 expression by cell sorting or by prolonged incubation with immunotoxin could reexpress CD33 at baseline levels and remained sensitive to immunotoxin. We conclude that humanized M195 conjugated to recombinant gelonin has antileukemic activity and should be considered for clinical testing in Phase I trials.

Antibodies, Monoclonal↗
PubMed1998

Chemotherapy immediately following autologous stem-cell transplantation in patients with advanced breast cancer.

Most patients relapse after high-dose chemotherapy (HDCT) with autologous stem-cell transplantation (ASCT) for metastatic breast cancer. Further chemotherapy immediately after hematopoietic recovery from ASCT is not given for fear of irreversibly damaging the newly engrafted stem cells. In a pilot chemoprotection trial, autologous CD34+ cells from patients with metastatic breast cancer were exposed to a replication-incompetent retroviral vector carrying MDR-1 cDNA and then reinfused after HDCT. Immediately on recovery, patients received multiple courses of escalating dose paclitaxel. All of the 10 patients tolerated reinfusion of modified cells without any toxicity and had myeloid engraftment within 12 days (range, 11-14). The bone marrow cells of three patients contained vector MDR-1-positive cells only at the time of the first course of posttransplant paclitaxel, indicating that the MDR-1 vector-modified cells had only short-term engrafting potential. A total of 83 courses of paclitaxel were administered starting at a median of 30 (range, 21-32) days from ASCT. The median dose of paclitaxel was 225 mg/m2 and the median interval between paclitaxel cycles of therapy was 21 (range, 20-41) days. Five of the six CR patients were able to receive all of the 12 courses of paclitaxel. Three patients who had achieved less than a complete response to the HDCT (2 patients) and partial response (1 patient) were converted to complete clinical responses during the 12 cycles of paclitaxel. No delayed toxicity or bone marrow failure was noted in these patients with a median follow-up of 2 years from ASCT. This is the first study of chemotherapy immediately after transplantation with autologous CD34+ cells. These data indicate that paclitaxel can be safely administered immediately after ASCT without any delayed toxicities. Paclitaxel given immediately after ASCT can further improve the response to pretransplant chemotherapy in patients with advanced breast cancer.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1997

Protease activation is required for glucocorticoid-induced apoptosis in chronic lymphocytic leukemic lymphocytes.

Recent work has demonstrated that glucocorticoids, nucleoside analogues, and other cancer chemotherapeutics induce apoptosis in chronic lymphocytic leukemia (CLL) cells. In this study, we investigated the involvement of protease activation in these responses using selective peptide inhibitors of the interleukin-1beta converting enzyme (ICE)/caspase family and a Ca2+-activated protease we recently implicated in thymocyte apoptosis. Apoptosis was associated with proteolytic cleavage of poly(adenosine diphosphate [ADP]-ribose) polymerase (PARP) and increased caspase protease activity, and cell-permeant caspase antagonists [zVAD(OMe)fmk and Boc-D(OBzl)cmk] blocked apoptosis in response to the glucocorticoid methylprednisolone or the nucleoside analogue fludarabine, indicating that caspase activation was required for these responses. However, a peptide-based inhibitor of the Ca2+-dependent lamin protease (zAPFcmk) also completely suppressed DNA fragmentation and the cleavage of lamin B1 . Strikingly, treatment of cells with zAPFcmk alone led to characteristic PARP cleavage, depletion of the precursor forms of two ICE family proteases (CPP32 and ICH-1), and phosphatidylserine exposure, suggesting that blockade of the lamin protease led to activation of the ICE family. Our results implicate the lamin protease as a target for Ca2+ during chemotherapy-induced apoptosis in CLL lymphocytes, and they identify a novel functional interaction between the protease and members of the ICE family.

Apoptosis↗
PubMed1997

Expression of epidermal growth factor receptor and human papillomavirus E6/E7 proteins in cervical carcinoma cells.

BACKGROUND: Epidermal growth factor receptor (EGF-R) proteins are highly expressed in many tumors, including those of the cervix. We have observed previously that the introduction of a transcription unit containing an antisense sequence for the E6/E7 genes of human papillomavirus (HPV) 18, along with a transcription unit containing a sense complementary DNA sequence for the wild-type retinoblastoma (Rb) gene, decreased the growth of human cervical carcinoma HeLa cells (HPV 18 positive) both in vitro and in vivo. To clarify the regulatory mechanisms by which this reduction in cell proliferation occurred, we studied the expression of EGF-R proteins in these cells. METHODS: Western blot and northern blot techniques were used to measure EGF-R expression, and a pulse-chase immunoprecipitation assay was used to measure the stability of EGF-R protein in HeLa cells and HeLa cells that had been transfected with the antisense E6/E7 or sense Rb sequences. Cell proliferation was measured by use of a tetrazolium-based colorimetric assay for numbers of viable cells. RESULTS: The introduction of sense Rb or antisense E6/E7 transcription units or a combination of these two transcription units into HeLa cells dramatically decreased the level of EGF-R proteins in these cells; EGF-R levels were not affected at the transcriptional level but at the post-transcriptional level. Addition of the anti-EGF-R-specific monoclonal antibody 225mAb to HeLa cells caused 53% (95% confidence interval = 44%-62%) growth inhibition. CONCLUSIONS: These results suggest that HeLa cervical carcinoma cells are dependent on EGF-R for proliferation and that changes in functional levels of the E6/E7 HPV proteins and endogenous Rb proteins may alter the growth rate of cervical cancer cell lines by reducing the stability of EGF-R at the post-transcriptional level.

Blotting, Northern↗
PubMed1997

Involvement of retinoic acid receptor-alpha-mediated signaling pathway in induction of CD38 cell-surface antigen.

Human leukocyte antigen CD38, a 45-kD single-chain, transmembrane glycoprotein, is a bifunctional ectoenzyme that participates in signal transduction pathways involved in the regulation of cell growth and differentiation. In this study, we demonstrate the nature of retinoid receptors involved in retinoic acid-induced expression of CD38 protein in the human myeloblastic leukemia cell line HL-60. We used a variant HL-60 cell line, HL-60R, in which retinoid receptor function has been abrogated by a trans-dominant negative mutation. We introduced the normal retinoic acid receptors (RAR)-alpha, -beta, and -gamma or retinoid X receptor (RXR)-alpha into HL-60R cells by retroviral vector-mediated gene transfer. Based on experiments using these cell lines and receptor-specific synthetic retinoids that preferentially bind to one of the RARs or RXRs, we conclude that RAR-alpha is involved in retinoid-induced CD38 expression in HL-60 cells. Further evidence included our demonstration that blocking of RAR-alpha with the antagonist Ro 41-5253 completely suppressed the retinoid-induced expression of CD38 mRNA transcript and the production of CD38 protein in HL-60 cells. Various tissues from transgenic mice that expressed an antisense construct of RAR-alpha lacked or produced very low levels of CD38. As expected, the tissues from transgenic mice contained 50% to 80% reduced levels of RAR-alpha. These results suggest that regulation of CD38 expression, both in vitro and in vivo, is under the direct control of RAR-alpha retinoid receptors.

ADP-ribosyl Cyclase↗