Noriomi Kuribayashi

Expression of Bcl-2 family of proteins in fresh myeloma cells

Members of the Bcl-2 family of proteins Second Department of Internal Medicine, Kumamoto University School of Medicine, Honjo 1-1-1, Kumamoto 860-8556, Japan Bcl-2, Bcl-XL, Bcl-Xs and Bax, are considered to play important roles in the regulation of apoptosis and drug resistance. To understand the significance of these proteins in fresh human myeloma cells, expression of Bcl-2 family of proteins was analyzed by Western blotting in 17 cases with multiple myeloma (MM) and three cases with plasma cell leukemia (PCL). Bcl-2 and Bcl-XL were found in 12 and nine samples, respectively. All PCL cases showed co-expression of Bcl-2 and Bcl-XL. Analysis of MM cases showed that Bcl-2 was preferentially expressed in samples from cases with early clinical stage while Bcl-XL tended to be expressed in samples from cases at advanced clinical stage. Bcl-XL was significantly expressed in tumor cells from cases with extramedullar lesions. There was no correlation between the expression levels of Bcl-2 or Bcl-XL and preceding chemotherapy. Expression of Bax was found in only one patient who had pleural effusion caused by invasion of myeloma cells and a high serum LDH level. Survival analysis revealed that there was no statistical significance in expression of Bcl-2 or Bcl-XL although Bcl-XL tended to be expressed in cases with poor prognosis. These findings indicate that expression of Bcl-2 family of proteins is heterogeneously regulated in fresh myeloma cells. Expression of Bcl-XL and Bcl-2 may correlate with extramedullar invasion and early stage of the disease, respectively. Absence of Bax in myeloma cells may contribute to low sensitivity of myeloma cells to anti-cancer agents since Bax is reported to mediate cytotoxicity of some anti-cancer drugs.

Detection of inducible nitric oxide synthase (iNOS) mRNA by RT-PCR in ATL patients and HTLV-I infected cell lines : clinical features and apoptosis by NOS inhibitor

Various tumors have been reported to express an inducible form of nitric oxide synthase (iNOS), and nitric oxide (NO) may affect the clinicopathological features of these tumors. Previously, Burkitt’s lymphoma and Epstein–Barr virus (EBV)-infected cells were shown to express iNOS constitutively at a low level. We analyzed iNOS expression by the reverse transcriptase-polymerase reaction method (RT-PCR) in eight HTLV-I-infected cell lines (five were ATL-derived lines and there were in vitro transformed lines), nine ATL patients (three were chronic, two were acute, and four were lymphoma type), and an HTLV-I-negative T cell line (CEM). In four ATL derived and in all three in vitro transformed cell lines, iNOS was expressed constitutively, but it was not expressed in CEM cells. Four out of nine ATL patients also showed iNOS expression. The expression of iNOS was found in all subtypes of ATL. Three of four iNOS-positive patients had infiltration of ATL cells to organs such as skin, lung, or liver. In NOS inhibitor (NG-monomethyl-L-arginine: L-NMMA)-containing medium, an iNOS-positive ATL cell line (K3T) showed growth inhibition and DNA ladder. Although only a limited number of patients was analyzed, our results suggest that NO may be involved in the invasive character of ATL cells. The NOS inhibitor can induce apoptosis in an ATL cell line, as it does in EBV-infected cell lines.

Expression of PRAD1/cyclin D1 in plasma cell malignancy: incidence and prognostic aspects

We analysed PRAD1/cyclin D1 expression in 20 patients with plasma cell malignancy by Northern analysis. 6/17 multiple myeloma patients and 3/3 plasma cell leukaemia patients showed PRAD1/cyclin D1 expression. This incidence appeared to be higher than the expected incidence based on previous studies. Southern analysis did not show rearrangement of the bcl‐1 region. Although there was no statistical difference, the PRAD1/cyclin D1 negative group showed a 1‐year survival of 81.8%, 3‐year survival of 45.5% and 5‐year survival of 22.7%, and those for the PRAD1/cyclin D1 positive group were 63.5%, 16.9% and 16.9%, respectively. Further study is required to determine whether PRAD1/cyclin D1 expression is a prognostic factor.

Multiple myeloma associated with serum amino acid disturbance and high output cardiac failure

We experienced a plasma cell leukemia (PCL) patient complicated with high output cardiac failure (HOCF), proved as his elevated cardiac index and pulmonary artery wedge pressure and decreased systemic vascular resistance index in a hemodynamic study. We found no possible causes of HOCF. Interestingly, HOCF was improved as PCL responded to intensive chemotherapy. On the other hand, he showed consciousness disturbance, and had frequent attacks of generalized seizure. His electroencephalogram showed slow waves, and a spike and wave complex. Hyperammonemia and abnormal amino acid distribution were also found. This abnormal serum amino acid distribution, especially elevated glycine level, was different from that seen in chronic liver failure, and he had no hepatic disease. After intensive chemotherapy, the serum ammonia level and glycine level decreased. In this patient, PCL seemed to be responsible for HOCF, hyperammonemia, and abnormal amino acid distribution. We experienced two more cases of multiple myeloma (MM) with HOCF, hyperammonemia, abnormal serum amino acid distribution, and consciousness disturbance of unknown origin. Those two cases showed slow waves in the electroencephalogram. Improvement was seen in their HOCF, hyperammonemia, and abnormal amino acid levels after chemotherapy. The possibility of MM as a cause of HOCF is discussed. Am. J. Hematol. 57:77–81, 1998.

Myelodysplastic syndrome following treatment of malignant melanoma with vincristine, ACNU, and dacarbazine.

Therapy‐related myelodysplastic syndrome is a rare adverse effect in melanoma patients elicited by chemotherapy. We report a case of myelodysplastic syndrome following treatment of malignant melanoma with alkylating agents. Peripheral blood showed a remarkable suppression of three cell lineages, and the bone marrow was slightly hypercellular. However, no morphological abnormalities were detected in the peripheral blood or the bone marrow, and chromosomal analysis was normal.

A Plasma Cell Leukemia Patient Showing Bialleic 14q Translocations: t(2;14) and t(11;14)

We report here an IgG/λ-type plasma cell leukemia patient showing bialleic 14q32 translocations. All immunoglobulins were suppressed in this patient, but a small amount of monoclonal IgG was detected by immunoelectrophoresis. Two cells of six peripheral blood mononuclear cells showed 46,XY,t(2;14)(q11;q32), i(8)(q10), t(11;14)(q13;q32), del(12)(q13.1) by karyotypic analysis. We confirmed the juxtaposition of IgH and PRAD1/Cyclin D1 genes by fluorescent in situ hybridization and overexpression of the PRAD1/Cyclin D1 gene, but Southern analysis showed no bcl-1 rearrangement. We analyzed the t(2;14)(q11;q32) using DNA fragments derived from childhood B-chronic lymphocytic leukemia cases bearing t(2;14)(p13;q32). Southern and Northern analyses demonstrated no alteration of these genes, indicating that this t(2;14) was different from that of childhood B-chronic lymphocytic leukemia. At the IgH loci, Southern analysis showed two rearranged bands and one germ-line band of JH. Cμ was deleted on one rearranged allele but remained on the other, suggesting that the chromosome translocation occurred after productive class switch recombination on the Cμ deleted allele.

Absence of Kaposi's sarcoma-associated herpesvirus (KSHV) in bone marrow cells from Japanese myeloma patients.

Absence of Kaposi’s sarcoma-associated herpesvirus (KSHV) in bone marrow cells from Japanese myeloma patients

Establishment and characterization of a CD95 (Fas/Apo-1)-negative myeloma cell line.

Although expression of CD95 (Fas/Apo-1) on myeloma cells has been reported, its significance is not clearly understood. We established a myeloma cell line, KHM-11ad (11ad), from a parental cell line, KHM-11, by collecting cells adhered to a plastic dish. KHM-11 cells have been reported to be positive for CD45 and CD95 (Fas/Apo1), and negative for a myelomonocytic antigen, CD13. Interestingly, CD95 was not detected in 11ad. Expression of CD45 was also significantly decreased in 11ad cells while expression of CD13 was detected in these cells. The growth rate of 11ad cells was 1.7 times lower than that of KHM-11 cells. Analysis of adhesion molecules showed that expression of VLA4 and CD44 was significantly suppressed in 11ad. The IC50 of melphalan (L-PAM) for 11ad cells was 50 times higher than that for KHM-11, indicating that 11ad is significantly refractory to L-PAM than KHM-11 cells. Induction of apoptosis by doxorubicin and cycloheximide was suppressed in 11ad cells compared with those in KHM-11 cells. Western blot analysis for Bcl-2 family of proteins showed that Bax was expressed at a 2.2 times lower level in 11ad cells than in KHM-11 cells while there was no difference in expression of Bcl-2, Bcl-Xs nor Bcl-XL. These results suggest that CD95-negative myeloma cells may have characteristics as follows: (1) slow proliferation; (2) low sensitivity to apoptosis; (3) low expression of VLA4, CD44 and Bax. Although these intraclonal variations were based on the findings of cell lines, these may reflect similar variations in vivo. The 11ad line may be a suitable model for analyzing intraclonal variation of myeloma cells.

Establishment of a monoclonal antibody to human myeloma cell: relation to chemotherapy and extramedullar infiltration

Resistance of myeloma cells to melphalan (L-PAM) is a serious problem. To investigate mechanisms of drug resistance, we generated a monoclonal antibody, clone O3, to melphalan-resistant myeloma cells, KHM-11R. Western blot analysis showed that molecular weight of O3 antigen was approximately 90 kDa. Expression of O3 antigen was approximately two times higher in KHM-11R than in parental melphalan sensitive cell line, KHM-11. O3 was preferentially expressed in plasma cell, B-cell, and monocytic cell lines, but not in T-cell lines. Analysis of bone marrow samples from myeloma patients revealed that 13 of 23 samples expressed O3 antigen at various levels, and that O3 antigen expression in patients correlate with preceding chemotherapy, advanced clinical stage and extramedullar invasion of myeloma cells. Furthermore, patients expressing O3 antigen at the time of diagnosis tended to have poor prognosis. The investigation of O3 antigen in myeloma cells will be useful to reveal the pathophysiology of extramedullar invasion and the mechanism of cell killing by melphalan.

Red blood cell volume (MCV) as a new prognostic factor of multiple myeloma

To the Editor: Anemia is known to be a factor prognostic of multiple myeloma and is used as a factor for staging (1). However, in our experience some myeloma patients with a higher MCV value, which is occasionally even above 100 fl, seem to respond to chemotherapy and survive longer, maintaining a good performance status despite their low hemoglobin concentration. To explain this observation, we analyzed 60 myeloma patients, whose onset and survival duration were clearly known, and evaluated their clinical stages, MCV and hemoglobin concentrations before treatment, as well as their survival duration. Untreated myeloma patients, consisting of 45 who had died and 15 who were alive, were classified retrospectively according to Durie and Salmons criteria (1). Sixteen, 13 and 31 patients were classified into stage 1,2, and 3, respectively. Patients were also classified into two groups according to their MCV value at diagnosis: group A (21 expired and 6 alive); MCV was greater than 97 fl, group B (24 expired and 9 alive); MCV was less than 97 fl (Normal range of MCV in our hospital: Male; 85.5-99.6 fl, Female; 83.5-99.1 fl. The MCV value was electrically analyzed by a Sysmex NE8000, Toa Medical Electrics Co. Ltd. Tokyo). Thus survival rate was evaluated by the Kaplan-Meier method. Group A showed a higher survival rate than group B at all observed points although the difference was not statistically significant according to the generalized Wilcoxons test (Fig. 1). The median survival duration in groups A and B was 40 and 26 months, respectively. Group A also showed a higher survival rate at all clinical stages than did group B (data not shown). This trend was also shown by the analysis of the expired cases. Analysis of the expired cases in group A and B at all stages, 21 and 24 cases, respectively, revealed a median survival duration of 46 and 20 months, respectively (p = 0.021 analyzed by Students t-test). These findings suggest that a high MCV correlates with longer survival. However, due to the very small size of this study, more cases should be analyzed to confirm this hypothesis. . . . . . . , .....,