Heat-shock protein 70 antagonizes apoptosis-inducing factor.

Heat-shock protein 70 (Hsp70) has been reported to block apoptosis by binding apoptosis protease activating factor-1 (Apaf-1), thereby preventing constitution of the apoptosome, the Apaf-1/cytochrome c/caspase-9 activation complex [1,2]. Here we show that overexpression of Hsp70 protects Apaf-1-/- cells against death induced by serum withdrawal, indicating that Apaf-1 is not the only target of the anti-apoptotic action of Hsp70. We investigated the effect of Hsp70 on apoptosis mediated by the caspase-independent death effector apoptosis inducing factor (AIF), which is a mitochondrial intermembrane flavoprotein [3,4]. In a cell-free system, Hsp70 prevented the AIF-induced chromatin condensation of purified nuclei. Hsp70 specifically interacted with AIF, as shown by ligand blots and co-immunoprecipitation. Cells overexpressing Hsp70 were protected against the apoptogenic effects of AIF targeted to the extramitochondrial compartment. In contrast, an anti-sense Hsp70 complementary DNA, which reduced the expression of endogenous Hsp70, increased sensitivity to the lethal effect of AIF. The ATP-binding domain of Hsp70 seemed to be dispensable for inhibiting cell death induced by serum withdrawal, AIF binding and AIF inhibition, although it was required for Apaf-1 binding. Together, our data indicate that Hsp70 can inhibit apoptosis by interfering with target proteins other than Apaf-1, one of which is AIF.

Hsp27 negatively regulates cell death by interacting with cytochrome c.

Mammalian cells respond to stress by accumulating or activating a set of highly conserved proteins known as heat-shock proteins (HSPs). Several of these proteins interfere negatively with apoptosis. We show that the small HSP known as Hsp27 inhibits cytochrome-c-mediated activation of caspases in the cytosol. Hsp27 does not interfere with granzyme-B-induced activation of caspases, nor with apoptosis-inducing factor-mediated, caspase-independent, nuclear changes. Hsp27 binds to cytochrome c released from the mitochondria to the cytosol and prevents cytochrome-c-mediated interaction of Apaf-1 with procaspase-9. Thus, Hsp27 interferes specifically with the mitochondrial pathway of caspase-dependent cell death.

Selective depletion of inducible HSP70 enhances immunogenicity of rat colon cancer cells.

Expression of inducible heat shock protein 70 (HSP70) in tumor cells has been proposed to enhance their immunogenicity. However, HSP70 has also been demonstrated to prevent tumor cell death, a key process for the development of tumor cell immunogenicity. In the present study, we investigated the influence of the HSP70 protein level on PRO colon cancer cell growth and immunogenicity in syngeneic BDIX rats and nude mice. These cells have a basal expression of HSP70 which can be substantially increased by heat shock. When injected subcutaneously in syngeneic animals, PRO cells do not induce any detectable immune response and give rise to progressive, metastatic and lethal tumors. Stable transfection of an anti-sense hsp70 cDNA in PRO cells (PRO-70AS cells) strongly decreased HSP70 expression and sensitized cell-free extracts to cytochrome c/dATP-mediated activation of caspases. Subcutaneous injection of PRO-70AS cells induced tumors that rapidly regressed in syngeneic rats while they grew normally in nude mice. Syngeneic rats injected with PRO-70AS cells became protected against a further challenge with PRO cells. The tumor-specific immune response induced by HSP70-depleted PRO-70AS cells was associated with an increased rate of cell death in vivo. These PRO-70AS cells were also more sensitive to NO-mediated, caspase-dependent, macrophage cytotoxicity in vivo. Altogether, these results indicate that reduced level of HSP70 expression in PRO- colon cancer cells results in the generation of a specific immune response by promoting cell death in vivo.

Significance of MDR1, MRP1, GSTpi and GSTmu mRNA expression in acute lymphoblastic leukemia in Indian patients.

Using, semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) in 167 patients of acute lymphoblastic leukemia (ALL) from India at different stages of the disease (presentation 125, remission 33, first relapse nine), MRP1 and GSTpi expression were significantly higher at relapse than presentation (P=0.03 and P=0.01, respectively) and remission (P=0.007 and P=0.003, respectively). MRP1, GSTpi and GSTmu were expressed simultaneously in several samples with significant association of expression levels (P=0.0001). Association with clinicopathological features included higher MDR1 expression with age >15 years (P=0.04) and higher MRP1, GSTpi, GSTmu expression with WBC counts >100x10(9)/l. In 71 patients (age <25 years), inability to achieve CR was associated with a significantly higher MDR1 mRNA expression (P=0.03) indicating a prognostic significance. However, relapse or shorter Event Free Survival was independent of mRNA expression levels of the four genes. In view of the increased mRNA expression of MRP1/GST at the time of relapse and an association with risk factors such as a high WBC count, further studies directed towards investigating the functional aspects of GSH/GST/MRP1 mediated drug transport are warranted.

Pattern of immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements in childhood acute lymphoblastic leukemia in India.

In 120 cases of acute lymphoblastic leukemia (median age 8 years), IgH chain gene was rearranged in 99% B-Cell Precursor (BCP) ALLs and 13% T-ALLs. One or the other TCR locus was rearranged not only in all T-ALLs, but also in 87% of BCP-ALLs. TCR-beta rearrangement in BCP-ALL was associated with a higher mean age at presentation (8.7 vs. 6.2 years, P=0.008), lower mean platelet counts (61.2x10(9)/l vs. 103.7x10(9)/l, P=0.003) and a poorer DFS (% cummulative survival 0 vs. 88.9+/-10.5, P=0.004). TCR-gamma rearrangement in T-ALL was associated with a higher mean WBC count (186.3x10(9)/l vs. 63. 4x10(9)/l, P=0.002). Also, the pattern of rearrangement of these genes appeared to be different from the West; viz. TCR-beta rearrangement in a higher proportion of BCP-ALLs (58%, 95% confidence intervals 45-69%), invariable deletion of Cgamma1 and only monoallelic rearrangement for TCR-delta locus. This repertoire of gene rearrangement may have a bearing on the poor treatment outcome reported previously from our geographic region.

Incidence, clinical characteristics and early treatment outcome in Indian patients of childhood acute lymphoblastic leukemia with ALL-1 gene rearrangement.

In a series of 185 patients (median age 7 years) of acute lymphoblastic leukaemia (ALL) from India, the overall incidence of ALL-1 gene rearrangement using the Southern blot technique was 11.4% (21/185). The incidence amongst the infants (age < or = 1 year, 70%) was significantly higher when compared to patients > 1 - < or = 10 years (7.4%, P = 0.00001) as well as > 10 years old (9.3%, P = 0.0001). ALL-1 gene rearrangement was associated with significantly higher WBC count (P = 0.01) and CD10 negativity (P = 0.00000001). Complete remission (CR) and relapse rates in 98 patients evaluable for response to therapy on a uniform therapy protocol was independent of ALL-1 gene status.

Detection of BCR-ABL transcripts in acute lymphoblastic leukemia in Indian patients.

Thirty-three patients with acute lymphoblastic leukemia (ALL) from India were studied for the presence of BCR-ABL chimeric transcripts, by a seminested cDNA-PCR. We report the presence of BCR-ABL chimeric transcripts in 4/17 (24%) children (under 15 years) and 3/16 (19%) adults (15-50 years). This is in sharp contrast to the published literature from the West where the presence of BCR-ABL has been reported in only 2-5% children and 35% adults. Whether the presence of BCR-ABL fusion mRNA, which is generally an attribute of ALL in adults and of poorer prognosis, may contribute to chemo-incurability in young Indian patients, remains to be seen, as a larger number of patients are studied for treatment outcome and survival on uniform therapy protocols.

A prospective study on the incidence of hepatitis B & C infections amongst patients with lymphoproliferative disorders.

Fifty one patients with acute lymphoblastic leukaemia (ALL) and non-Hodgkins lymphoma (NHL) undergoing chemotherapy were studied prospectively to determine the incidence, aetiology and natural course of hepatitis. Of 51 patients (31 NHL and 20 ALL), 22 developed hepatitis. Hepatitis B (IgM anti HBc positive) was the cause in 11 patients (50%), hepatitis C in 4 patients, and septicaemia and cytotoxic drugs in 3 patients each. Malignant infiltration of the liver was the cause in the remaining 1 patient. Hepatitis was predominantly (75%) anicteric. Mean duration of hepatitis was 21 days. Of 51 patients, 21 acquired hepatitis B and/or C virus infection. They had received 6.4 (+/- 3.4) units of packed red cells and 5.3 (+/- 11) units of platelet concentrate as compared to 3.4 (+/- 4.8) units of red cells and 5.3 (+/- 12.1) units of platelet concentrate received by those who did not acquire virus infection (P < 0.05 for packed red cells). Only transient stoppage of chemotherapy was necessary following development of hepatitis and most of the patients who developed hepatitis could complete their chemotherapy schedule. None of the patients who developed viral B or C infection cleared the infection. We conclude that there was a high incidence of hepatitis B and C infection amongst patients with lymphoproliferative disorders with an increased carrier rate. Transfusion was a major risk factor for such infections.

AKT is a therapeutic target in myeloproliferative neoplasms.

The majority of patients with BCR-ABL1-negative myeloproliferative neoplasms (MPN) harbor mutations in JAK2 or MPL, which lead to constitutive activation of the JAK/STAT, PI3K and ERK signaling pathways. JAK inhibitors by themselves are inadequate in producing selective clonal suppression in MPN and are associated with hematopoietic toxicities. MK-2206 is a potent allosteric AKT inhibitor that was well tolerated, including no evidence of myelosuppression, in a phase I study of solid tumors. Herein, we show that inhibition of PI3K/AKT signaling by MK-2206 affected the growth of both JAK2V617F- or MPLW515L-expressing cells via reduced phosphorylation of AKT and inhibition of its downstream signaling molecules. Moreover, we demonstrate that MK-2206 synergizes with ruxolitinib in suppressing the growth of JAK2V617F-mutant SET2 cells. Importantly, MK-2206 suppressed colony formation from hematopoietic progenitor cells in patients with primary myelofibrosis and alleviated hepatosplenomegaly and reduced megakaryocyte burden in the bone marrows, livers and spleens of mice with MPLW515L-induced MPN. Together, these findings establish AKT as a rational therapeutic target in the MPNs.

A novel clofarabine bridge strategy facilitates allogeneic transplantation in patients with relapsed/refractory leukemia and high-risk myelodysplastic syndromes.

Patients with relapsed/refractory leukemias or advanced myelodysplastic syndrome (MDS) fare poorly following allogeneic hematopoietic cell transplant (HCT). We report prospective phase II study results of 29 patients given clofarabine 30 mg/m(2)/day i.v. × 5 days followed immediately by HCT conditioning while at the cytopenic nadir. A total of 15/29 patients (52%) were cytoreduced according to pre-defined criteria (cellularity <20% and blasts <10%). Marrow cellularity (P<0.0001) and blast% (P=0.03) were reduced. Toxicities were acceptable, with transient hyperbilirubinemia (48%) and gr3-4 infections (10%). In all, 28/29 proceeded to transplant; 27 received ATG or alemtuzumab. Post HCT, 180 day non-relapse mortality (NRM) was 7% (95% confidence interval (CI): 1-21), relapse was 29% (95% CI: 13-46) and OS was 71% (95% CI: 51-85), comparing favorably to published data for high-risk patients. Two-year graft vs host disease incidence was 40% (95% CI: 21-58) and 2 year OS was 31% (95% CI: 14-48). Disease at the nadir correlated with inferior OS after HCT (HR=1.22 for each 10% marrow blasts, 95% CI: 1.02-1.46). For AML/MDS patients, there was a suggestion that successful cytoreduction increased PFS (330 vs 171 days, P=0.3) and OS (375 vs 195 days, P=0.31). Clofarabine used as a bridge to HCT reduces disease burden, is well tolerated, and permits high-risk patients to undergo HCT with acceptable NRM. Late relapses are common; thus, additional strategies should be pursued. NCT-00724009.

Overexpression of survivin initiates hematologic malignancies in vivo.

Survivin is an inhibitor of apoptosis protein family member that has an essential role in cellular proliferation as a component of the chromosome passenger complex. Survivin is highly expressed in embryos and in proliferating adult tissues, but it is not expressed in most differentiated cells. During tumorigenesis, however, survivin expression is dramatically upregulated. Although many studies have shown that survivin is required for cancer cells, the extent to which survivin contributes to the initiation of tumors is unknown. Here we show that transgenic mice that overexpress survivin in hematopoietic cells are at an increased risk of hematologic tumors. In examining how survivin might contribute to tumorigenesis, we observed that hematopoietic cells engineered to overexpress survivin are less susceptible to apoptosis. We conclude that survivin may promote tumorigenesis by imparting a survival advantage to cells that acquire additional genetic lesions.

Heat shock proteins: endogenous modulators of apoptotic cell death.

The highly conserved heat shock proteins (HSPs) accumulate in cells exposed to heat and a variety of other stressful stimuli. HSPs, which function mainly as molecular chaperones, allow cells to adapt to gradual changes in their environment and to survive in otherwise lethal conditions. The events of cell stress and cell death are linked and HSPs induced in response to stress appear to function at key regulatory points in the control of apoptosis. HSPs include antiapoptotic and proapoptotic proteins that interact with a variety of cellular proteins. Their expression level can determine the fate of the cell in response to a death stimulus, and apoptosis-inhibitory HSPs, in particular HSP27 and HSP70, may participate in carcinogenesis. This review summarizes apoptosis-regulatory function of HSPs.

Expression of the multidrug resistance-associated protein (MRP) mRNA and protein in normal peripheral blood and bone marrow haemopoietic cells.

We studied the expression of multidrug resistance-associated protein (MRP) in normal haemopoietic cells from peripheral blood and bone marrow. The MRP mRNA levels were estimated by RT/PCR and in situ hybridization (ISH) assay, and the protein levels by flow cytometry. 21 samples of peripheral blood and 21 samples of bone marrow (11 normal bone marrow donors, 10 patients in complete remission after chemotherapy for large cell lymphoma or acute myeloid leukaemia) were analysed. In peripheral blood the mean MRP mRNA level in CD3+ cells was statistically higher than in the other cells (3-fold by the methods used). The levels of MRP in CD3+ varied from one individual to another (4.5-34.8 units by RT/PCR and 5-23 grains/cell by ISH); however, this was proportional to the variation in all the cell lineages of same individual (r = 0.84). In bone marrow the mean MRP levels of the various cell lineages (including CD34+) were similar to the basal level in HL60 cells. Individual expression levels were again variable; however, there was no difference between untreated normal bone marrow and post chemotherapy normal bone marrow. MRP protein expression was determined by flow cytometry with the monoclonal antibody MRPm6. The CD4+ lymphocytes exhibited a higher MRP protein expression than the other cell lineages, including CD8+ cells. There was a good correlation between the three methods used (RT/PCR and ISH, P = 0.0001, r = 0.87; RT/PCR and flow cytometry, P = 0.0001, r = 0.85; ISH and flow cytometry, P = 0.002, r = 0.67).

Expression of genes implicated in multidrug resistance in acute lymphoblastic leukemia in India.

In order to investigate the phenomenon of multidrug resistance as a possible mechanism for poor response to treatment in patients with acute lymphoblastic leukemia (ALL) from India, a series of 32 cases of de novo untreated ALLs were analyzed by a cDNA-PCR approach to estimate the relative mRNA levels of the MDR-associated genes encoding MDR1, MRP, GSTpi, and GSTmu. The expression of beta2 microglobulin served as an internal standard. Quantifiable transcripts were observed in 20 patients for MRP, in 5 for MDR1, in 24 for GSTpi, and in 19 for GSTmu. The values ranged from undetectable to 132% of the control A549 cell line for MRP, undetectable to 49% of the HL60/DNR control cell line for MDR1, undetectable to 268% of A549 control cell line for GSTpi, and undetectable to 247% of A549 control cell line for GSTmu mRNA. Increased MRP levels were associated with increased GSTpi and GSTmu levels (p<0.01 for both), and increased levels of MDR1 were associated with increased GSTpi levels (p<0.05). The present observations showed no correlation between the MDR1 and MRP values with treatment outcome, in terms of either achieving a complete remission or predilection to early relapse. In view of some recent studies that envisage MRP as an energy-dependent pump involved in the efflux of GSH conjugates, the simultaneous up-regulation of transcription of all these genes might well be part of an integrated detoxification response that has been switched on after exposure to an environmental stress.

Identification of tumor-infiltrating macrophages as the killers of tumor cells after immunization in a rat model system.

Immunization can prevent tumor growth, but the effector cells directly responsible for tumor cell killing in immunized hosts remain undetermined. The present study compares tumor grafts that progress in naive syngeneic rats with the same grafts that completely regress in hosts preimmunized with an immunogenic cell variant. The progressive tumors contain only a few macrophages that remain at the periphery of the tumor without direct contact with the cancer cells. These macrophages do not kill tumor cells in vitro. In contrast, tumors grafted in immunized hosts and examined at the beginning of tumor regression show a dramatic infiltration with mature macrophages, many of them in direct contact with the cancer cells. These macrophages are strongly cytotoxic for the tumor cells in vitro. In contrast to macrophages, tumor-associated lymphocytes are not directly cytotoxic to the tumor cells, even when obtained from tumor-immune rats. However, CD4(+) and CD8(+) T cells prepared from the regressing tumors induce tumoricidal activity in splenic macrophages from normal or tumor-bearing rats and in macrophages that infiltrate progressive tumors. These results strongly suggest that the main tumoricidal effector cells in preimmunized rats are macrophages that have been activated by adjacent tumor-immune lymphocytes.