Taizo Tasaka

A novel treatment strategy targeting Aurora kinases in acute myelogenous leukemia

The Aurora kinases play an important role in chromosome alignment, segregation, and cytokinesis during mitosis. Aberrant expression of these kinases occurs in solid tumors and is associated with aneuploidy and carcinogenesis. We found in this study that Aurora kinase A and B were aberrantly expressed in a variety of types of human leukemia cell lines (n = 15, e.g., PALL-1, PALL-2, HL-60, NB4, MV4-11, etc.), as well as freshly isolated leukemia cells from individuals with acute myelogenous leukemia (n = 44) compared with bone marrow mononuclear cells from healthy volunteers (n = 11), as measured by real-time PCR. ZM447439 is a novel selective Aurora kinase inhibitor. The compound induced growth inhibition, caused accumulation of cells with 4N/8N DNA content, and mediated apoptosis of human leukemia cells as measured by thymidine uptake, cell cycle analysis, and annexin V staining, respectively. Especially profound growth inhibition occurred with the PALL-1 and PALL-2 cells, which possess wild-type p53 gene. In contrast, ZM447439 did not inhibit clonogenic growth of myeloid committed stem cells harvested from healthy normal volunteers. Taken together, inhibition of Aurora kinases may be a promising treatment strategy for individuals with leukemia. [Mol Cancer Ther 2007;6(6):1851–7]

Decreased expression of p33ING1 mRNA in lymphoid malignancies

The ING1 is a newly cloned putative tumor‐suppressor gene, which is involved in the p53 signaling pathway. We found decreased expression of ING1 mRNA in 4 of 5 T‐cell lines and 5 of 11 B‐cell lines including two Burkitt lymphomas and two myelomas. These observations suggest that decreased ING1 expression might play an important role in the development or progression of some lymphoid tumors. Polymerase chain reaction‐SSCP and sequencing analyses found neither point mutations nor small deletions in the ING1 gene, suggesting that decreased expression is due to transcriptional or post‐transcriptional mechanisms. Am. J. Hematol. 62:118–119, 1999.

Analysis of the p16INK4A, p15INK4B and p18INK4C genes in multiple myeloma

To study the structural integrity of the cyclin‐dependent kinase inhibitors known as INK4A (p16), INK4B (p15) and INK4C (p18) in multiple myeloma, we examined 20 primary myeloma samples (including one case of plasma cell leukaemia) using polymerase chain reaction–single strand conformation polymorphism, and 17 samples were examined by Southern blot analysis. The plasma cell leukaemia sample had homozygous deletions of the p15 and p16 genes (6%). One myeloma case had a p15 gene homozygous deletion (6%) with an intact p16 gene. This sample also had a p18 homozygous deletion, suggesting that the deletion of both genes may be important in either the development or progression of myeloma. No point mutations of these INK4 genes were found in the 20 samples. This is the first report that indicates that deletions of p15, p16 and p18 genes occur in some individuals with multiple myeloma (2/17 cases).

Methylation of the p16INK4A gene in multiple myeloma

The p16INK4A (p16) binds to both cyclin D‐CDK4 and cyclin D‐CDK6 and inhibits the progression of the cell cycle from G1 to S phase. Loss of expression of this protein can occur by several mechanisms including structural alterations. Recent studies have suggested that the loss of expression of p16 can occur by hypermethylation of the gene. The methylation status of the p16 gene in multiple myeloma was examined in three myeloma cell lines (U266, RPMI8226 and IM9) and 16 primary myeloma samples using methylation‐specific polymerase chain reaction (MSP). The U266 and RPMI8226 cell lines contained a completely methylated p16 gene and the IM9 line had a partially methylated p16 gene. Identical results were obtained by another polymerase chain reaction (PCR)‐based methylation assay system as well as Southern blotting after using a methylation‐sensitive restriction enzyme. The U266 cell line expressed no p16, and the IM9 had weak expression as determined by reverse transcript (RT‐)PCR. The U266 cells began to express, and IM9 increased the accumulation of, the p16 RNA after treatment with the demethylating agent 5′‐aza‐2‐deoxycytidine (10−6–10−5 M). This suggested that the levels of methylation of the p16 gene detected by the MSP technique correlated with the regulation of transcription of this gene. Examination of the primary myeloma samples showed that eight of 16 (50%) contained a methylated p16 gene. We have previously found that alterations of the p16 gene, such as deletions and point mutations, are rare in primary multiple myeloma; none of the 16 samples included in this study had p16 gene alterations. Our results suggest that methylation of the p16 gene may contribute to the development and/or progression of multiple myeloma.

Lenalidomide induces cell death in an MDS-derived cell line with deletion of chromosome 5q by inhibition of cytokinesis.

Myelodysplastic syndromes (MDS) are a group of hematopoietic stem cell disorders characterized by refractory cytopenias and susceptibility to leukemic transformation. On a subset of MDS patients with deletion of the long arm of chromosome5 (del(5q)), lenalidomide exerts hematological and cytogenetic effects, but the underlying pharmacological mechanisms are not fully understood. In this study, we have investigated the in vitro effects of lenalidomide on an MDS-derived cell line, MDS-L, which carries del(5q) and complex chromosome abnormalities. We found that the growth of MDS-L cells was specifically suppressed mainly by apoptosis, and in addition, multinucleated cells were frequently formed and finally died out in the presence of lenalidomide. Time-lapse microscopic observation and the DNA ploidy analysis revealed that lenalidomide does not affect DNA synthesis but inhibits cytokinesis of MDS-L cells. The gene expression profile showed decreased expression of M phase-related genes such as non-muscle myosin heavy-chain 10, polo-like kinase 1, aurora kinase B, citron kinase and kinesin family member 20A(KIF20A). Interestingly, KIF20A is located at 5q31. These data contribute to the understanding of action mechanisms of lenalidomide on MDS with del(5q) and complex abnormalities.

A novel treatment strategy targeting polo-like kinase 1 in hematological malignancies

Objectives: Polo-like kinase1 (PLK1) belongs to the family of serine/threonine kinases and plays an important role in centrosome maturation, bipolar spindle formation, and cytokinesis during mitosis. We found in this study that PLK1 was aberrantly highly expressed in a variety of human leukemia cell lines (n=20), as well as, freshly isolated leukemia cells from individuals with acute myelogenous leukemia (n=50) and acute lymphoblastic leukemia (n=15) compared with bone marrow mononuclear cells from healthy volunteers (n=13) (acute myelogenous leukemia, P=0.016; acute lymphoblastic leukemia, P=0.008), as measured by real-time RT–PCR. Downregulation of PLK1 by a small interfering RNA in NB4 acute myelogenous leukemia cells inhibited their proliferation. GW843682X is a novel selective PLK1 inhibitor. The compound-induced growth inhibition, caused accumulation of cells in the G2/M phase of the cell cycle and mediated apoptosis of human leukemia cells. Pre-treatment of cells with the caspase inhibitor Z-VAD-FMK attenuated the action of GW843682X in leukemia cells, indicating the involvement of the caspase pathway in the PLK1 inhibitor-mediated apoptosis. Furthermore, we found that the PLK1 inhibitor synergistically potentiated the growth inhibition and apoptosis of leukemia cells when combined with tubulin-depolymerizing agent vincristine. Taken together, targeting PLK1 may be a promising treatment strategy for individuals with leukemia.

Allelotyping of acute myelogenous leukemia: loss of heterozygosity at 7q31.1 (D7S486) and q33-34 (D7S498, D7S505).

Loss of a whole chromosome 7(-7) or a deletion of the long arm of chromosome 7 del(7q) occurs frequently in many types of primary cancers including cases of acute myelogenous leukemia (AML). We analyzed for loss of heterozygosity (LOH) of chromosome arm 7q in 26 AML cases using a set of 15 microsatellite markers in order to begin to determine the location of putative tumor suppressor genes (TSG) important to this disease. Seven samples (27%) showed LOH at one or more loci on chromosome 7q. We identified the smallest commonly deleted regions to be at 7q31.1 (D7S486) and 7q33-34 (D7S498, D7S505) suggesting that alterations of a TSG in each region have an important role in de novo AML.

Microsatellite instability during the progression of acute myelocytic leukaemia

We studied microsatellite instability (MSI) at the onset and during progression of 17 individuals with acute myelocytic leukaemia (AML). These included two cases of M0, eight with M1 and seven with M4, according to the FAB classification. The DNA from diagnostic, remission and relapsed stages of their disease was analysed at 69 loci. Two MSI were found in the diagnostic and remission phase paired samples (12%), and eight MSI were identified in six of the relapsed phase samples (35%). These results indicate that mismatch repair errors such as MSI are unimportant at the onset of AML, but might have importance during the progression of the disease.

CD8+, CD56+ (natural killer-like) T-cell lymphoma involving the small intestine with no evidence of enteropathy: clinicopathology and molecular study of five Japanese patients.

The present study reports five CD8+, CD56+ (natural killer (NK)‐like) T‐cell lymphomas involving the small intestine without evidence of enteropathy, from Japan. Three were intestinal T‐cell lymphoma. The site of origin of the other two was not definitive. Four of five patients underwent emergency operation because of intestinal perforation. The small intestines of these patients had multiple ulcerative lesions with or without demarcated tumors. Histologically, the lymphoma cells were monomorphic or slightly pleomorphic and displayed epitheliotropism of varying degrees. Lymphoma cells of all patients shared the common phenotype: CD3+, CD4−, CD5−, CD8+, CD56+, CD57−, T‐cell intracellular antigen‐1+, granzyme B+. In contrast to nasal/nasal type NK‐cell lymphomas, they had clonal rearrangement of T‐cell receptor(TCR) genes and were negative for EBV‐encoded RNA. Immunohistochemistry and genetics suggested that three cases were of αβT‐cell origin and two cases were of γδT‐cell origin. There was no evidence of enteropathy in any patient. The cases followed a clinically aggressive course with a frequent involvement of lung. According to the classification based on the recent genetic studies of European enteropathy‐type intestinal T‐cell lymphoma (ETL), the present cases could be classified as type 2 ETL.

Infrequent microsatellite instability during the evolution of the myelodysplastic syndrome to acute myelocytic leukemia

Microsatellites are highly polymorphic, short-tandem repeat sequences dispersed throughout the genome. Instability of these repeat sequences at multiple gentic loci may result from mismatch repair errors and occur in hereditary nonpolyposis colorectal cancer and several other sporadic cancers, including chronic myelocytic leukemia as it progresses to blastic crisis. We investigated whether genetic instability occurred as myelodysplasia progressed to acute myelocytic leukemia. To this end, we studied microsatellite instability in 20 patients with myelodysplastic syndrome (MDS). These included five patients with refractory anemia (RA), three with refractory anemia with ringed sideroblast (RARS), nine with refractory anemia with excess blasts (RAEB) and three with chronic myelomonocytic leukemia (CMML). All of these patients transformed to acute myelocytic leukemia (AML) of various subtypes: three patients with M1, 11 with M2 and six patients with M4 (according to FAB classification). The DNA from both the MDS and AML phases of their disease was analyzed at 16 loci, and only four microsatellite instabilities were found in the 240 paired samples (1.6%) analyzed. These results indicate that mismatch repair errors such as microsatellite instability are not important in the evolution of MDS to AML.