Establishment of five human myeloma cell lines

Summary Five human myeloma cell lines, KMM-1, KMS-5, KMS-11, KMS-12- PE, and KMS-12-BM, have been established at Kawasaki Medical School since 1980. As the KMS-12-PE and KMS-12-BM lines were obtained from the same patient, these five cell lines have been derived from four patients with multiple myeloma. The five myeloma cell lines are stably growing at present in RPMI 1640 medium supplemented with 10% fetal bovine serum. They can also grow in a defined culture medium without serum. That these cell lines were, human myeloma cells was confirmed by the following findings. Ultranstructually, all five cell lines showed features characteristic of plasma cells. KMM-1 and KMS-11 cells secreted lambda and kappa chains into the culture medium, respectively, but the other cell lines produced no immunoglobulins. KMM-1 expressed cytoplasmic lambda antigen, KMS-5 showed cytoplasmic delta, and KMS-11 expressed surface kappa, whereas KMS-12-PE and KMS-12-BM cells showed no surface or cytoplasmic immunoglobulins. Regarding reaction with a monoclonal plasma cell antibody (PCA-1), four of the five lines were positive, the exception being KMS-5. Another monoclonal antibody (CD38), which also recognizes plasma cells, reponded to KMM-1, KMS-12-PE, and KSM-12-BM. KMS-5 cells expressed acute lymphoblastic leukemia antigens (CALLA). These data suggest that such lines as KMM-1, KMS-11, KMS-12-PE, and KMS-12-BM represent later stages of B-cell differentiation, and that KMS-5 represents a relatively early stage of B-cell differentiation. All the cell lines lacked Epstein-Barr virus nuclear antigen, showed abnormal karyotypes of human origin, and differed from each other in the isozyme patterns examined. Only KMS-5 was tumorigenic when transplanted subcutaneously into nude mice.     

Identification of cellular differentiation-dependent nuclear factors that bind to a human gene for thymidylate synthase.

Three nuclear factors were identified that interact with sequences in the 5'-upstream region of the human thymidylate synthase gene. Two of these factors interact with a sequence around the initiation codon of the thymidylate synthase gene. The amounts of these two factors changed dramatically as human promyelocytic leukemia HL-60 cells differentiated into macrophage-like cells by the treatment with 1,25-dihydroxyvitamin D3. The change was closely correlated with the decrease in the amount of thymidylate synthase mRNA during the differentiation. These findings suggest that the specific nuclear factors are involved in the regulation of the expression of human thymidylate synthase gene during the differentiation of HL-60 cells.     

A restriction fragment length polymorphism (RFLP) locus of rat (Rattus norvegicus) linked to theCs-1 locus of rat linkage group XIII

A novel restriction fragment length polymorphism (RFLP) in inbred rats was revealed by Southern blot analysis with a clone arbitrarily chosen from a rat genomic library as a probe. A clone named alpha 403 showed interstrain variations in the length of the EcoRI and HindIII fragments. The EcoRI fragments were either 0.7 or 3 kb, those of HindIII were either 4.5 or 5 kb, and three types were identified as combinations of those fragments in 20 inbred rat strains. These types segregated in backcross progeny as codominant alleles. The locus for the RFLP was thus named A403. Analysis of linkage between the RFLP locus and 13 other loci reveal that the A403 locus was closely linked to the Cs-1 locus (15 +/- 5.2%), which belongs to rat linkage group XIII.     

Unusual Branching in the Seedlings of Lotus japonicus--Gibberellins Reveal the Nitrogen-sensitive Cell Divisions within the Pericycle on Roots

We studied the effects of several plant-growth regulators onthe induction of nodule-like structures on roots of Lotus japonicus,which has been proposed as a candidate for a leguminous plantfor molecular genetic analysis. Contrary to our expectations,the addition of gibberellin A₃ (GA₃) at concentrations of 10^-4M to 10^-4 M resulted in the formation of nodule-like structureson roots when seedlings were plated on nitrogen-free Fahraeusagar medium. GA₄ also induced such outgrowths but was less activethan GA₃. Application of an inhibitor of auxin transport, N-(1-naphthyl)-phthalamicacid (NPA) and of kinetin, which have been reported to inducepseudonodules in other legumes, had no effect on L. japonicus.Microscopic observations showed that GA₃-induced nodule-likestructures were caused by cell divisions within the pericycleon the roots. In addition, the outgrowths elicited by GA₃ couldbe completely suppressed by the addition of 15 mM potassiumnitrate or ammonium nitrate. These results show that the pericyclecells of the roots of L. japonicus are specifically sensitiveto gibberellins and that potential for cell division might bemodulated by nitrogen compounds. We also examined the effectsof ancymidol and uniconazole [S-3307; (E)-1-(4-chIorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-yl)-1-penten-3-ol],two synthetic plant-growth retardants. Both compounds at 3 x10^-5 M significantly increased the number of stunted lateralroots. The unusual branching could not be counteracted by theexogenous addition of GA₃ but by 10^-6 M brassinolide. We discussthe physiological role of brassinolide in the initiation oflateral roots. (Received August 4, 1995; Accepted March 11, 1996)     

The Excessive Production of Indole-3-Acetic Acid and Its Significance in Studies of the Biosynthesis of This Regulator of Plant Growth and Development

Because of the importance of indole-3-acetic acid (IAA) in thegrowth and development of plants, extensive studies of the biosynthesisof IAA have been performed during the four decades since thediscovery of IAA as a plant hormone. The pathway for the biosynthesisof IAA in plants remains, however, to be unelucidated, eventhough studies within the past decade have revealed unexpectedaspects of such biosynthesis. By contrast, two pathways to IAAhave been characterized in bacteria at the molecular level:the indole-3-acetamide (IAM) pathway (L-tryptophan     

Effect of β-alanyl-L-histidinato zinc on differentiation of osteoblastic MC3T3-El cells: Increases in alkaline phosphatase activity and protein concentration

The effect of -alany-L-histidinato zinc (AHZ) on bone cell function was investigated in osteoblastic MC3T3-E1 cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus AHZ (10^–7–10^–5 M) or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5 M) produced a remarkable increase of alkaline phosphatase activity and protein concentration in osteoblastic cells. Thus increases were seen with the prolonged cultivation (12–21 days). With the culture of 1, 3 and 12 days, the effect of AHZ (10^–6 M) to increase alkaline phosphatase activity and protein concentration was more intensive than the effect of zinc sulfate, (10^–6 M). The AHZ effects were completely abolished by the presence of cycloheximide (10^–6 M), indicating that AHZ stimulates protein synthesis in the cells. The present study suggests that AHZ has a stimulatory effect on cell differentiation, and that this effect is partly involved on protein synthesis in osteoblastic cells.     

Distribution of Vacuolar H+-Pyrophosphatase and a Membrane Integral Protein in a Variety of Green Plants

Vacuolar membranes isolated from several species including fernand moss exhibited pyro-phosphate-dependent H⁺ transport activity.On immunoblot analysis, H⁺ -pyrophosphatase was detected inthe vacuolar membranes. A membrane integral protein of 23,000daltons was not found in the membranes of Chara, Conocephalum,or Kalanchoë. Thus, H⁺-pyrophosphatase may be a universalenzyme among green plants, but the 23-kDa protein is not a commonprotein of central vacuoles. (Received September 10, 1993; Accepted November 29, 1993)     

Stimulatory effect of β-alanyl-L-histidinato zinc on cell proliferation is dependent on protein synthesis in osteoblastic MC3T3-E1 cells

The effect of -alanyl-L-histidinato zinc (AHZ) on bone metabolism was investigated in osteoblastic MC3T3-El cells. Cells were cultured for 3 days at 37°C in a CO₂ incubator in plastic dishes containing -modified minimum essential medium supplemented with 10% fetal bovine serum. After the cultures, the medium was exchanged for that containing 0.1% bovine serum albumin plus various concentrations of AHZ or other reagents, and the cells were cultured further for appropriate periods of time. The presence of AHZ (10^–7–10^–5M) stimulated the proliferation of cells. AHZ (10^–6 and 10^–5M) increased deoxyribonucleic acid (DNA) content in the cells with 48hr-culture. This increase was completely blocked by the presence of cycloheximide (10^–6M) or hydroxyurea (10^–3M). Also, the presence of cycloheximide (10^–6M) completely inhibited the AHZ (10^–5M)-induced increase in the proliferation of cells. Meanwhile, parathyroid hormone (10^–7M), estrogen (10^–9M) and insulin (10^–M) significantly increased cellular DNA content. However, these hormonal effects clearly lowered in comparison with that of AHZ (10^–5M). Dibutyryl cyclic AMP (10^–4M) and zinc sulfate (10^–5M) did not cause a significant increase in cellular DNA content. The present results support the view that AHZ has a direct specific proliferative effect on osteoblastic cellsin vitro and that this effect is dependent on protein synthesis.