Search

We utilized three tiers of screening to identify novel therapeutic agents for pancreatic cancers. First, we analyzed 14 pancreatic cancer cell lines against a panel of 66 small-molecule kinase inhibitors and dasatinib was the most potent. Second, we performed RNA expression analysis on 3 dasatinib-resistant and 3 dasatinib–sensitive pancreatic cancer cell lines to profile their gene expression. Third, gene profiling data was integrated with the Connectivity Map database to search for potential drugs. Thioridazine was one of the top ranking small molecules with highly negative enrichment. Thioridazine and its family members of phenothiazine including penfluridol caused pancreatic cancer cell death and affected protein expression levels of molecules involved in cell cycle regulation, apoptosis, and multiple kinase activities. This family of drugs causes activation of protein phosphatase 2 (PP2A). The drug FTY-720 (activator of PP2A) induced apoptosis of pancreatic cancer cells. Silencing catalytic unit of PP2A rendered pancreatic cancer cells resistant to penfluridol. Our observations suggest potential therapeutic use of penfluridol or similar agent associated with activation of PP2A in pancreatic cancers. [ABSTRACT FROM AUTHOR]

Presents a letter as contribution to the research about disease progression in adult T-cell leukemia/lymphoma.

EB1089, a novel 1,25-dihydroxyvitamin D3 analogue, has been known to have potent antiproliferative properties in a variety of malignant cells both in vitro and in vivo. In the present study, we analysed the effect of EB1089 on NCI-H929 human myeloma cells. EB1089 inhibited cell growth of NCI-H929 and efficiently induced the G1 phase arrest of the cell cycle in a dose-dependent manner. We could also detect apoptosis in NCI-H929 cells exposed to EB1089 (1 x 10-7 M for 72 h) using the sub-G1 group of the cell cycle by FACS and annexin V binding assays. Induction of apoptosis by EB1089 was associated with down-regulation of the Bcl-2 protein without change of the Bax protein. Regarding caspase activity, which plays a crucial role in apoptosis, EB1089-treated NCI-H929 cells revealed an increased activity of caspase 3 protease accompanied by degradation of the PARP protein in a dose- and time-dependent manner. In addition, EB1089 caused the down-regulation of p44 extracellular signal-related kinase (ERK) activity and up-regulation of the p38 kinase activity during apoptosis of NCI-H929 cells. These results suggest that EB1089 inhibits growth of NCI-H929 cells via G1 cell cycle arrest as well as apoptosis by activating p38 kinase and suppressing ERK activity.

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.

Ataxia-telangiectasia (A-T) is a multisystem recessive disease characterized by cerebellar ataxia, oculocutaneous telangiectasias, immunodeficiency and increased risk of cancer. The ATM gene, responsible for A-T, was recently cloned at human chromosome band 11q22-23, a region of frequent alterations in childhood acute lymphoblastic leukaemia (ALL). Children with A-T frequently develop T-ALL. We investigated 18 T-ALL samples for ATM mutations and loss of heterozygosity (LOH) at the ATM locus. No mutations of ATM were found within the coding region in the 18 T-ALL samples, and LOH at the ATM locus was detected in three. The ATM gene appears to be an infrequently altered tumour suppressor gene in childhood T-ALL.

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.

Microsatellite instability (MSI) is thought to represent a defect of the DNA mismatch repair system which has been implicated in the tumourigenesis of several human malignancies. We investigated MSI in acute/ lymphomatous adult T-cell leukaemia (ATL: n=22) using 54 highly polymorphic dinucleotide short-tandem repeat sequences. The corresponding control DNA from each individual was obtained from the peripheral blood in either chronic phase (n=5) or when complete remission was achieved (n=17). 10/22 (41%) patients had MSI, six of whom showed MSI in multiple loci; four loci had MSI in multiple samples. The incidence of MSI in ATL was found to be higher than in other haematological malignancies, indicating MSI as a feature of ATL, which may be involved in the progression of the disease.

p18INK4C, a cyclin-dependent kinase inhibitor, is a homologue of p15INK4B and p16INK4A which are frequently altered in a variety of malignancies. We searched for structural alterations of the p18INK4C gene in 44 adult T-cell leukaemias (ATLs), 101 non-Hodgkin's lymphomas (NHLs), two polyclonal B-cell proliferations, seven ATL cell lines and seven leukaemia/lymphoma cell lines, by Southern blot and polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) analyses. No genomic alterations of the p18INK4C gene were found in any of the samples. By RT-PCR, p18INK4C was not expressed in three of five ATL cell lines, whereas it was expressed in all the non-ATL leukaemia/lymphoma cell lines. Tax did not inhibit the expression of p18INK4C in tax-expressing Jurkat cells.

We studied microsatellite instability (MSI) at the onset and during progression of 17 individuals with acute myelocytic leukaemia (AML). These included two cases of MO, 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.

Microsatellite instability (MSI) has been considered to represent the defect of DNA mismatch repair systems and has been implicated in the tumourigenesis of several human malignancies. To investigate the possible presence of microsatellite instability in childhood acute lymphoblastic leukaemia (ALL), we examined 48 primary ALL samples. Instability was determined at 85 different microsatellite loci localized to 12 different chromosome arms. Microsatellite instability was detected in five (10%) samples. Interestingly, the instability was found at chromosomal regions associated with frequent alterations. Two samples had instability at the microsatellite marker within the TEL gene on chromosome arm 12p. Two other samples had instability at a microsatellite marker close to CDKN2/p16 on 9p; one of these samples had a homozygous deletion at 9p21. The fifth sample had instability at the microsatellite marker on 6q, which we have found is a frequent region of loss of heterozygosity in childhood ALL. Taken together, instability was rare in childhood ALL, but was localized to the three most frequently deleted chromosome regions in childhood ALL, suggesting that localized microsatellite instability may identify a fragile chromosomal region which could result in alteration of surrounding target genes and lead to leukaemia.

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).

A B-cell lymphoma cell line, designated KML-1, was established from pleural effusion of a patient with non-Hodgkin's lymphoma of large-cell type. The lymphoma arose in the pelvis and ran an aggressive clinical course. Chromosome analysis of the cell line exhibited a complex karyotype including the loss of chromosome 18. To evaluate the molecular events in the cell line that may be associated with the development of the lymphoma, we investigated the expression and/or alterations of several classes of human genes, including oncogenes, tumor suppressor genes, and cytokine genes. The expression of the DCC (deleted in colorectal cancer) gene, located on the chromosome 18q21, was extremely reduced in KML-1 cell line, as compared with that in a normal spleen tissue and other 4 lymphoma cell lines by the reverse transcription-polymerase-chain-reaction (RT-PCR) method. This finding suggests that inactivation of the DCC gene might play a role in the pathogenesis of the case of lymphoma.

Generation of superoxide may be a key step in the cytotoxicity mediated by tumour necrosis factor (TNF); cells that cannot produce oxygen radicals might be resistant to TNF. Myeloid haemopoietic cells from patients with chronic granulomatous disease (CGD) cannot produce a large burst of oxygen radicals; therefore we examined the ability of TNF to inhibit clonal growth of myeloid haemopoietic cells from patients and carriers with several types of CGD. Mononuclear light-density cells from the peripheral blood of 13 CGD patients (11 patients with defects of gp91-phox and two with p47-phox), five gp91-phox carriers and 10 normal volunteers were cultured with the appropriate growth factor and TNF in methylcellulose. As expected, TNF (0.001-100 ng/ml) inhibited colony formation of myeloid cells of normal volunteers in a dose-dependent manner. In contrast, clonal growth of myeloid cells of CGD patients was resistant to inhibition by TNF < or = 100 ng/ml. As expected, the effects of TNF on erythroid clonogenic cells, which are not capable of producing an oxygen burst, and the action of TGF-beta on clonal growth of myeloid cells, were similar in both the individuals with CGD and the normal volunteers. In X chromosome-linked female carriers of CGD (gp91-phox deficiency), TNF showed an intermediate cytotoxicity on clonal growth of myeloid cells, and analysis of NBT reduction demonstrated that the colonies derived from myeloid cells deficient in gp91-phox were resistant to TNF and those derived from the myeloid cells expressing gp91-phox were inhibited in their proliferation by TNF. This study shows for the first time that myeloid haemopoietic cells from patients with CGD are relatively resistant to the growth-inhibiting effects of high concentrations of TNF.

Haemopoietic cells with pluripotent capabilities, were grown in soft-gel cultures from the peripheral blood of a glucose-6-phosphate dehydrogenase (G6PD) electrophoretic A,B heterozygous female with chronic myelocytic and granulocytic progeny. Only type B isoenzyme of G6PD was found in mixed colonies, in mature neutrophils, erythrocytes, and committed granulocyte and isoenzymes of G6PD. This study provides direct evidence that a pluripotent haemopoietic stem cell is involved in the malignant clone in CML.

We studied the effect of the H2-receptor antagonists, cimetidine and metiamide, on in vitro myeloid colony formation by bone marrow cells from seven normal volunteers and two patients with a cimetidine-associated neutropenia. A cimetidine concentration of 500 microgram/ml produced 50% inhibition of normal granulocyte-macrophage colony formation, and 1000 microgram/ml of cimetidine completely suppressed proliferation. The inhibitory effect of metiamide occurred at lower concentrations: 50% inhibition at 250 microgram/ml and 95% inhibition at 350 microgram/ml. Cimetidine had a similar inhibitory effect on colony formation by recovery marrow from the two patients with cimetidine-associated neutropenia. Treatment of autologous and allogeneic marrows with patients' acute phase sera and cimetidine failed to show evidence of antibody-mediated suppression of granulopoiesis. Our results indicate that at sufficiently high concentrations, cimetidine and metiamide inhibit human bone marrow myeloid colony formation in vitro. These findings are consistent with the hypothesis that H2-receptor antagonists may produce neutropenia in a dose-related fashion by injury to granulocytic progenitor cells in vivo.

The effect of amphotericin B on human and murine hematopoiesis was studied in vitro using assays for erythroid and myeloid colony-forming cells. Amphotericin consistently inhibited colony formation by erythroid and granulocyte-monocyte progenitor cells. Clear effects were observable at amphotericin concentrations of 1.0 microgram/ml, and concentrations of 2.0 microgram/ml caused approximately 50-60% inhibition of cloning of both murine and normal human bone marrow. These data suggest that amphotericin, in concentrations achieved in therapy, can impair hematopoiesis by a direct effect on precursor cells.

Phorbol esters can induce differentiation into macrophage-like cells in the two human myeloid leukaemia cell lines HL-60 and KG-1. We investigated the requirement for DNA synthesis in this differentiation process. We found that phorbol ester-induced differentiation of blast cells into cells with the morphologic, histochemical, and phagocytic characteristics of macrophages did not require 3H-thymidine incorporation. Our results indicate that phorbol ester induced macrophage differentiation does not require DNA synthesis.

We have investigated the mechanism of splenic irradiation-induced granulocytopenia in two patients with myelofibrosis and marked splenomegaly. Serial assays were performed for circulating granulocyte-monocyte progenitors capable of colony formation in vitro (CFU-C). For comparison, similar studies were performed on two patients receiving whole brain irradiation for glioma. Splenic irradiation caused a significant decrease in circulating CFU-C in the myelofibrosis patients. There was no decrease in circulating CFU-C in the brain-irradiated patients. No radiation-induced humoral inhibitor of granulopoiesis and no increased CFU-C radiosensitivity could be demonstrated in the myelofibrosis patients. These observations, taken together with previous data on splenic blood flow and pooling, suggest that the major mechanism of irradiation-induced granulocytopenia in myelofibrosis is destruction of proliferating precursor cells in the splenic tissue and sinusoids.