Hematopoietic cytokines enhance Chk1-dependent G2/M checkpoint activation by etoposide through the Akt/GSK3 pathway to inhibit apoptosis.

Hematopoietic cytokines play crucial roles in regulation of cell cycle progression and apoptosis of hematopoietic cells. However, the effects of cytokines on cellular responses to chemotherapeutic agents and the mechanisms involved have remained elusive. Here we report that erythropoietin or IL-3 promotes G2/M arrest and prevents apoptosis induced by the topoisomerase II inhibitor etoposide in murine hematopoietic 32D cells and human leukemic UT7 cells. Erythropoietin or IL-3 significantly enhanced etoposide-induced activation-specific phosphorylation of Chk1, a checkpoint kinase that inhibits Cdc2 activation by Cdc25 phosphatases, and led to the inhibition of Cdc2 kinase activity with the persistent inhibitory phosphorylation on Tyr15. The inhibitory Cdc2 phosphorylation and G2/M block by etoposide were enhanced or inhibited by overexpression of Chk1 or by the specific Chk1 inhibitor SB218078, respectively. The G2/M arrest induced by etoposide was also enhanced or inhibited by expression of a constitutively activated or dominant-negative Akt mutant, respectively. Furthermore, SB216763 or LiCl, a specific inhibitor for the GSK3 kinase inhibited by Akt, enhanced the Chk1 phosphorylation and G2/M arrest by etoposide. These results indicate that hematopoietic cytokines protect etoposide-treated cells from DNA damage-induced apoptosis by promoting, through the PI3K/Akt/GSK3 signaling pathway, G2/M checkpoint that is dependent on Chk1-mediated inhibition of Cdc2.

SOCS-3 inhibits IL-12-induced STAT4 activation by binding through its SH2 domain to the STAT4 docking site in the IL-12 receptor beta2 subunit.

IL-12 promotes the proliferation of T cells as well as NK cells and plays a critical role in induction of the Th1 differentiation. IL-12 mediates its biological activities through activation of the receptor-associated JAK family kinases and STAT4, which is recruited to phosphorylated Tyr-800 in the human IL-12 receptor beta2 subunit (IL-12Rbeta2). Here we demonstrate that suppressor of cytokine signaling-3 (SOCS-3) is also recruited to IL-12Rbeta2 by the interaction involving the SOCS-3 SH2 domain and phosphorylated Tyr-800 in IL-12Rbeta2. Furthermore, SOCS-3, but not its SH2 domain-defective mutant, inhibited the IL-12-induced activation of DNA-binding and transcriptional activities of STAT4. These results suggest that SOCS-3, expressed at high levels in Th2 cells, plays an inhibitory role in STAT4-mediated IL-12 signaling by binding to the STAT4 docking site in IL-12Rbeta2, thus raising a possibility that SOCS-3 may play a role in regulation of Th differentiation.

T-cell prolymphocytic leukemia with der(11)t(1;11)(q21;q23) and ATM deficiency.

T-cell prolymphocytic leukemia (T-PLL) is a rare mature T-cell malignancy and is similar to a mature T-cell leukemia seen in some patients with ataxia telangiectasia, which is a recessive hereditary chromosomal instability syndrome caused by mutations of the ataxia telangiectasia mutated (ATM) gene located on 11q23. Intriguingly, recent studies have strongly implicated ATM in the pathogenesis of T-PLL as a tumor suppressor gene, because biallelic inactivation of ATM is frequently observed in this disease; however, translocations involving 11q23 have rarely been reported in T-PLL. We report here a case of T-PLL with der(11)t(1;11)(q21;q23). Southern blot analysis did not reveal any abnormality of ATM, nor of MLL, which is also located on 11q23 and is involved in t(1;11)(q21;q23) in acute myelomonocytic leukemia. Northern blot analysis further showed that the ATM transcript of normal size is expressed in the leukemic cells at a level higher than that in normal peripheral blood lymphocytes. Western blot analysis, however, revealed that expression of ATM in the leukemic cells is much lower than that in normal lymphocytes. These results imply that translation of the ATM transcript is impaired or that the ATM protein is highly unstable in the leukemic cells, thus suggesting the presence of nucleotide changes in both alleles.

Aberrant expression of the LHX4 LIM-homeobox gene caused by t(1;14)(q25;q32) in chronic myelogenous leukemia in biphenotypic blast crisis.

Chromosomal aberrations observed in addition to the Philadelphia chromosome in chronic myelogenous leukemia (CML) are likely to be involved in disease progression to the blast crisis. We describe here a t(1;14)(q25;q32) as an additional chromosomal aberration in a patient with CML in biphenotypic blast crisis. By use of long-distance inverse polymerase chain reaction (PCR), we cloned the chromosomal breakpoint and revealed that the immunoglobulin heavy chain gene is fused near its Emu enhancer region to the 5' region of the LHX4 LIM-homeobox gene, whose expression is restricted to the central nervous system. By use of quantitative real-time reverse-transcription PCR, we found that the LHX4 mRNA is expressed at high levels in the patient's leukemic cells and in an acute lymphoblastic leukemia (ALL) cell line. The aberrant expression of the LHX4 gene by the t(1;14)(q25;q32) has very recently been reported in a case of ALL, thus, representing a rare, but recurrent genetic abnormality of possible importance in leukemogenesis.

[Late appearance of Philadelphia chromosome with the p190 BCR/ABL chimeric transcript in acute myelogenous leukemia progressing from myelodysplastic syndrome].

We report a late appearance of the Philadelphia chromosome (Ph) with the p190 BCR/ABL chimeric transcript in a 69-year-old patient with acute myelogenous leukemia (AML) that had evolved from myelodysplastic syndrome (MDS). In July 1997, the patient was found to have pancytopenia caused by refractory anemia with excess of blasts, which evolved into AML in 4 months. The leukemic cells were positive for CD13, CD14, CD33, and HLA-DR and had a normal karyotype. The patient achieved a complete remission after combination chemotherapy. However, his leukemia relapsed in November 1999, with the appearance of leukemic cells positive for CD7, CD13, CD34, and HLA-DR with a 46, XY, add (18) (p11) karyotype. The patient failed to achieve the second remission after several courses of intensive chemotherapy. When the number of blastic cells, showing the same surface phenotypes, in the peripheral blood increased drastically in April 2000, chromosomal analysis of leukemic cells revealed a 46, XY, t(9;22) (q34;q11), add(18)(p11) karyotype. The fusion of the BCR and ABL genes was confirmed by fluorescence in situ hybridization analysis, and the reverse transcription-polymerase chain reaction analysis further revealed the presence of the p190 BCR/ABL chimeric transcript. The appearance of the Ph chromosome in the course of MDS transforming to AML is very rare and may be correlated to the disease progression.

Fusion of MLL and MSF in adult de novo acute myelomonocytic leukemia (M4) with t(11;17)(q23;q25).

The MLL gene at chromosome band 11q23 is frequently rearranged and fused to partner genes in acute leukemias. Previously, the MSF gene, also called AF17q25, has been cloned as a fusion partner of the MLL gene in therapy-related or infant acute myelogenous leukemias with t(11;17)(q23;q25). MSF belongs to the septin family of proteins, which includes other MLL fusion partners, hCDCrel1 and Septin 6, and has also been implicated in the pathogenesis of human ovarian tumor and murine T-cell lymphoma. We describe here a 64-year-old man with de novo acute myelomonocytic leukemia (French-American-British subtype M4) with t(11;17)(q23;q25). His leukemia was successfully induced into a first remission, which, however, lasted only briefly. A second remission was never attained, and the patient died of sepsis 16 months after the diagnosis of leukemia. Examination of his leukemic cells at diagnosis revealed an MLL gene rearrangement, by Southern blotting, and an MLL-MSF fusion transcript, by the reverse transcriptase polymerase chain reaction (RT-PCR) method. Sequence analysis of the RT-PCR product further revealed that MLL exon 5 was fused in-frame to MSF exon 3. Further clinical and molecular analyses of acute leukemias with the MLL-MSF transcript may shed more light on the clinical characteristics and molecular mechanisms of the MLL-septin type leukemias.

[Hypercalcemia mediated by parathyroid hormone-related protein in the blastic phase of chronic myelogenous leukemia].

A 45-year-old man with chronic myelogenous leukemia (CML) in the accelerated phase was admitted to our hospital because of lower back pain and hypercalcemia. On admission, he was confused and found to have massive splenomegaly. The hypercalcemia and splenomegaly improved significantly after administration of incadronate, hydroxyurea, vincristine and prednisolone. Splenomegaly recurred after cessation of the chemotherapy, and examination of the peripheral blood showed 31% blasts, positive for both CD13 and CD33, on which basis myeloid blastic transformation was diagnosed. Vindesine, cytarabine and prednisolone were administered, and the splenomegaly improved again. On admission, when the patient's serum calcium level was 16.0 mg/dl, his serum parathyroid hormone-related protein (PTHrP) level was elevated to 118.3 pmol/l. Furthermore, RT-PCR analysis revealed that the patient's CML cells expressed PTHrP mRNA, and a high level of PTHrP was detected in the supernatant of cultured mononuclear cells derived from the patient's peripheral blood. These findings indicated that the hypercalcemia was due to production of PTHrP by the leukemic cells. Several cases of PTHrP. mediated hypercalcemia associated with CML have been reported previously, and are reviewed here.