Relationships between DNA methylation and expression in erythrocyte membrane protein (band 3, protein 4.2, and beta-spectrin) genes during human erythroid development and differentiation.

Red cell membrane proteins are sequentially expressed during erythroid development and differentiation. Spectrins have already been synthesized in early erythroid precursors such as pronormoblasts, and band 3 (B3) appears at nearly the same stage. Protein 4.1 appears next, followed by protein 4.2 (P4.2) at the very late erythroblast stage. The methylation states of the promoter 5'-CG-3' sites are known to be linked to the regulation of promoter function by modulating DNA-protein interactions and the structure of chromatin. Hence, the genes for B3, P4.2, and beta-spectrin (beta-SP) appear to be suitable models to study the relationship between methylation of promoter 5'-CG-3' sites and the sequential expression of genes during human erythroid development and differentiation. We have examined methylation profiles in the promoter regions of the genes (ELB42, EPB3, and SPTB) for the human erythroid membrane proteins P4.2, B3, and beta-SP by applying the bisulfite genomic sequencing method. Our results demon strate the following: (1) The promoter regions of EPB3 and ELB42 are extensively methylated in DNA from human peripheral blood mononuclear cells, but the SPTB promoter is totally unmethylated. (2) During erythroid differentiation, DNA methylation patterns change as follows: (a) ELB42 is unmethylated in DNA from erythroid-committed blastic cells, such as the human cell lin UT-7/EPO, but is methylated in erythroblasts from peripheral blood burst-forming unit erythroid (BFU-E) in the second phase of the liquid-culture method. Messenger RNA (mRNA) from ELB42 is first detected in early erythroblasts, and P4.2 is expressed in late erythroblasts. (b) In contrast, EPB3 is consistently methylated in UT-7/EPO cells and in cultured erythroblasts from BFU-E from human peripheral blood. B3 mRNA and protein are already expressed in early erythroblasts. (c) SPTB remains unmethylated in human DNA from UT-7/EPO cells and from cultured erythroblasts. These results document the diversity of the reactions of human promoter sequences to the modulating influence of DNA methylation. Whereas the human SPTB promoter conforms to expectations in that it is unmethylated and fully active throughout erythroid development, high levels of promoter methylation correlate with promoter activity for the EPB3 and ELB42 genes during their sequential activation in erythrocyte differentiation.

DNA methylation in promoter regions of red cell membrane protein genes in healthy individuals and patients with hereditary membrane disorders.

The methylation state of 5'-CG-3' sites is known to be linked to the regulation of promoter function by modulating DNA-protein interactions and to the structure of chromatin. As part of a project to determine methylation patterns in the human genome, we examined the methylation profiles of several genes for human erythroid membrane proteins: ELB42 (protein 4.2), EPB3 (band 3), SPTB gene (beta-spectrin), and ANK1 (ankyrin). The bisulfite protocol of the genomic sequencing method was applied. The number of 5'-CG-3' dinucleotides was the most abundant in SPTB and ANK1, much less in EPB3, and the least in ELB42. In the DNA of peripheral blood mononuclear cells from healthy individuals, the promoter regions of EPB3 and ELB42 were extensively methylated, but the SPTB and ANK1 promoters were totally unmethylated. We also investigated methylation profiles in peripheral blood mononuclear cells from patients with red cell membrane diseases, such as complete protein 4.2 deficiency due to ELB42 mutations, hereditary spherocytosis with EPB3 mutations, and hereditary elliptocytosis with SPTB mutations. The DNA methylation states in these genes of erythroid cells, which we obtained at the second phase of the 2-phase liquid culture of erythroid precursor cells in the peripheral blood, were essentially identical or very similar to those of peripheral blood mononuclear cells. In disease states, the DNA methylation profiles of these red cell membrane protein genes were essentially not different from those in healthy individuals (statistically not significant).

Expression of angiogenic factors including VEGFs and the effects of hypoxia and thalidomide on human myeloma cells.

Angiogenic factors are major causes of tumor progression in hematological malignancies, particularly multiple myeloma, as well as solid tumors. The introduction of thalidomide as an anti-angiogenic agent in myeloma treatment has demonstrated the importance of angiogenic factors in the progression of myeloma. However, the direct effects of angiogenic factors, particularly VEGFs, hypoxia, and thalidomide, on myeloma cells are not been documented. In this study, we demonstrate increased expression and production levels of VEGF in myeloma compared to non-myelomatous hematological lines, resistance to hypoxia and enhancement of VEGF-A production by hypoxia in myeloma, and direct growth inhibition of myeloma cells due to apoptosis and G1 arrest caused by TNFalpha upregulation induced by thalidomide. These findings may encourage the clinical use of anti-angiogenic agents for their cytostatic effects and the prevention of progression.

Synergistic growth inhibition of YM529 with biologic response modifiers (BRMs) in myeloma cells.

Bisphosphonates (BPs) are effective in the management of bone disease in patients with multiple myeloma. Recent reports have suggested that they may also have an antitumor activity. YM529 is a new synthetic BP with more than 1000 times the bone resorption inhibitory activity of pamidronate. To clarify the direct effects of YM529 on myeloma cells, the cell proliferation and cell cycle perturbation were analyzed using 12 myeloma cell lines established in our laboratory. The growth inhibition was dose dependent. The cells accumulated in [2n<<4n] of the cell cycle and subsequently formed an apoptotic sub-G1 fraction. Combined treatment with all-trans retinoic acid, thalidomide, or interferon-alpha enhanced the growth inhibitory effects of YM529 on these cells. However, there were no remarkable effects of YM529 on the messenger RNA expression for angiogenic factors, cell cycle regulators, or cytokines related to myeloma cells. These results indicate that YM529 is beneficial not only to bone lesions but also for its direct antitumor effects on myeloma cells.

Multiple myeloma complicated by autoimmune hemolytic anemia.

A 57-year-old man was admitted with severe anemia and hypergamma globulinemia. After a diagnosis of multiple myeloma and autoimmune hemolytic anemia was made, chemotherapy rapidly decreased the M-protein level and improved his anemia with normalization of the direct Coombs test. The immunoglobulin binding to the patient's red cells was immunoglobulin G kappa chain like the myeloma M-protein. However, monoclonal immunoglobulin G derived from short-term culture of the patient's bone marrow mononuclear cells did not bind to a panel of red cells. Therefore, the relationship between the M protein produced by his myeloma cells and hemolysis remained unclear.

[Pneumonitis with a bronchiolitis obliterans organizing pneumonia-like shadow in a patient with human herpes virus-6 viremia after allogeneic bone marrow transplantation].

We report the case of a 42-year-old male who underwent allogeneic bone marrow transplantation (BMT) for acute myelogenous leukemia, and then developed pneumonitis with a bronchiolitis obliterans organizing pneumonia (BOOP)-like shadow. When he came with exertional dyspnea four months after BMT, the chest X-ray and CT findings disclosed bilateral infiltration, and remarkable elevation of his serum KL-6 level, a monitoring marker for disease activity in interstitial lung disease. Although organizing pneumonia (OP) was revealed by a transbronchial lung biopsy, no pathogen was detected in bacterial, fungal and routine viral cultures or by direct cytological examinations using bronchoalveolar lavage (BAL) specimens. Since human herpes virus-6 (HHV-6) was detected in BAL specimens by the polymerase chain reaction (PCR), a diagnosis of a pneumonitis-like BOOP shadow related to HHV-6 was made, and he was treated with methylprednisolone and ganciclovir (GCV). Although there was a relapse of his OP 1.5 months later, with re-elevation of his serum KL-6 level, continuous administration of GCV led to disappearance of HHV-6 in BAL specimens assayed by PCR, in association with normalization of the serum KL-6 level. HHV-6 should be considered as a cause of unexplained pneumonitis in BMT recipients, and KL-6 is useful for monitoring the pneumonitis status in these patients.

[Acute myelogenous leukemia associated with severe esophageal stricture after chemotherapy].

A 41-year-old woman with relapsed acute myelogenous leukemia was treated twice with idarubicin hydrochloride and cytarabine. She developed a 6-cm-long stricture in the lower esophagus 12 days after re-induction therapy. Although she had preceding candida infection, it is suspected that her stricture was caused by mucosal damage due to chemotherapeutic agents. This case suggests that the possibility of esophageal stricture should be considered in patients with swallowing disturbance after intensive chemotherapy.

[Seven patients with stage I and II primary testicular lymphoma].

Seven patients with stage I and II primary testicular lymphoma (PTL) have been treated since 1990 to the present at Kawasaki Medical School. All patients, whose median age was 56 yrs, had initially complained of swelling of the scrotal contents. The lesions were on the right in two patients, on the left in five, and no patient had bilateral lesions. The histological diagnosis was diffuse large B-cell type in all patients. Five patients were classified as Ann Arbor stage I, and two at stage II. After high-orchiectomy for resection of tumor, two patients received chemotherapy alone, with a combination of chemotherapy and irradiation of the contralateral testis in the remaining five. Complete remission was achieved in all seven patients, but relapse occurred later in one. As it is recognized that, even in localized stage or low risk group PTL patients, the relapse rate in the central nervous system (CNS) and contralateral testis is quite high, chemotherapy, prophylactic CNS treatment and radiation of the contralateral testis after tumor resection should be included in the management of PTL.

Interleukin 10 abolishes the growth inhibitory effects of all-trans retinoic acid on human myeloma cells.

Recently, it was disclosed that all-trans retinoic acid (ATRA) inhibits myeloma cell growth by downregulating the interleukin 6 (IL-6)/IL-6 receptor (IL-6R) auto/paracrine loop, and upregulating p21/Cip1 cyclin-dependent kinase inhibitor (CDK-I), thereby inducing apoptosis with a decrease in Bcl-2 protein expression. To elucidate and generalize the effects of ATRA on the proliferation and cellular biology of myeloma cells, 12 human myeloma cell lines established in our laboratory were utilized. Two out of the 12 lines showed enhanced growth on supplementation of ATRA and were characterized by IL-10 production, downregulation of membrane Fas and reduced upregulation of p21/Cip1 CDK-I message. These characteristics may prove important for the clinical use of ATRA and should be considered before starting ATRA therapy for myeloma.

Diagnostic significance of serum soluble transferrin receptors in various anemic diseases: the first multi-institutional joint study in Japan.

Serum soluble transferrin receptor (sTfR) has been reported to be higher in patients with iron deficiency or with elevated erythropoiesis. In the present study, serum sTfR was measured in various anemic diseases and their clinical significance was examined in a multi-institutional joint study. Serum sTfRs in patients with the following anemic diseases were markedly higher than those in normal healthy adults: non-treated iron deficiency anemia (IDA) (9.13 +/- 7.04 mg/l, n = 52, p < 0.0001), anemia of chronic disorders (ACD) (3.45 +/- 1.38 mg/l, n = 20, p < 0.0001), hemolytic anemia (HA) (5.57 +/- 3.26 mg/l, n = 17, p < 0.0001), and myelodysplastic syndrome (MDS) (4.03 +/- 2.83 mg/l, n = 20, p < 0.0001). There were significant differences between IDA and ACD (p < 0.0001), between aplastic anemia (AA) (1.58 +/- 1.26 mg/l, n = 16) and MDS (p < 0.001), and between AA and MDS with refractory anemia (MDS-RA) (4.16 +/- 3.40 mg/l, n = 9) (p < 0.02). In patients with chronic renal failure (CRF), serum sTfR levels and serum sTfR/log serum ferritin ratios (sTfR/F index) were compared in the two classified groups according to Muirhead's criteria, as IDA and non-IDA groups with or without recombinant human erythropoietin (rHuEPO) treatment. Significantly high levels of both serum sTfR (p < 0.0001) and the sTfR/F index (p < 0.0001) were observed in IDA without rHuEPO treatment. Especially in CRF with rHuEPO treatment, the sTfR/F index showed marked elevation in the IDA group (p < 0.0001) compared with serum sTfR (p < 0.001), indicating more diagnostic efficacy of the sTfR/F index for CRF with IDA. In conclusion, the serum sTfR concentration is a useful diagnostic tool for discrimination between IDA and ACD, and between AA and MDS-RA, and for the detection of iron deficiency in CRF patients in the Japanese population.