Influence of Oral Anaerobic Bacteria on Hematopoietic Stem Cell Transplantation Patients: Oral Mucositis and General Condition.

OBJECTIVE: Oral mucositis (OM) caused by infection facilitated by myelosuppression and immunosuppression can be controlled through oral care. We investigated changes in oral anaerobic bacterial flora during the onset of OM with hematopoietic stem cell transplantation (HSCT). METHODS: This study included 19 patients who underwent HSCT. All received professional oral care before initiating the preparative regimen. We assessed OM, oral health and obtained microbial samples from the oral cavity during 5 assessment points: before initiating the preparative regimen; the day before HSCT (day 1); and at 7, 14, and 30 days after HSCT. Microbial species were identified by using a mass spectrometer. RESULTS: The number of patients with serious OM increased initially after HSCT and decreased thereafter. Many Streptococcus species were identified before HSCT, but these gradually decreased and were replaced by coagulase-negative staphylococci. An increase in Candida species after HSCT and the identification of Enterococcus species were significantly associated with OM. Nutritional status recovery and prognosis were significantly worse in patients who developed OM. CONCLUSIONS: To the best of our knowledge, this study is the first which shows that anaerobic bacteria were identified in patients' oral flora before and after HSCT by using a mass spectrometer. These results indicate that Enterococcus species and Candida species may have been associated with OM. OM affected the patients' improvement in nutritional status and their prognosis. We concluded that it is important to provide more complete oral care instructions and interventions to prevent these bacterial infections.

KPT-330 has antitumour activity against non-small cell lung cancer.

BACKGROUND: We investigated the biologic and pharmacologic activities of a chromosome region maintenance 1 (CRM1) inhibitor against human non-small cell lung cancer (NSCLC) cells both in vitro and in vivo. METHODS: The in vitro and in vivo effects of a novel CRM1 inhibitor (KPT-330) for a large number of anticancer parameters were evaluated using a large panel of 11 NSCLC cell lines containing different key driver mutations. Mice bearing human NSCLC xenografts were treated with KPT-330, and tumour growth was assessed. RESULTS: KPT-330 inhibited proliferation and induced cell cycle arrest and apoptosis-related proteins in 11 NSCLC cells lines. Moreover, the combination of KPT-330 with cisplatin synergistically enhanced the cell kill of the NSCLC cells in vitro. Human NSCLC tumours growing in immunodeficient mice were markedly inhibited by KPT-330. Also, KPT-330 was effective even against NSCLC cells with a transforming mutation of either exon 20 of EGFR, TP53, phosphatase and tensin homologue, RAS or PIK3CA, suggesting the drug might be effective against a variety of lung cancers irrespective of their driver mutation. CONCLUSIONS: Our results support clinical testing of KPT-330 as a novel therapeutic strategy for NSCLC.

Expression of transferrin receptor 2 in normal and neoplastic hematopoietic cells.

Iron is essential for cell proliferation, heme synthesis, and a variety of cellular metabolic processes. In most cells, transferrin receptor-mediated endocytosis is a major pathway for cellular iron uptake. Recently, transferrin receptor 2 (TfR2), another receptor for transferrin, was cloned. High levels of expression of TfR2 messenger RNA (mRNA) occur in the liver, as well as in HepG2 (a hepatoma cell line) and K562 (an erythroid leukemia cell line). In this study, TfR2 mRNA expression was analyzed in hematological cell lines, normal erythroid cells at various stages of differentiation, and leukemia and preleukemia cells. High levels of TfR2 expression occurred in all of the erythroid cell lines that were examined. Erythroid-specific expression of TfR2 protein in bone marrow cells was confirmed by immunohistochemical staining. Expression of TfR2 mRNA was high in normal CD34(+) erythroid precursor cells, and levels decreased during erythroid differentiation in vitro. Levels of expression of TfR2-alpha mRNA were significantly higher in erythroleukemia (M6) marrow samples than in nonmalignant control marrow samples. In addition, relatively higher levels of TfR2-alpha mRNA expression occurred in some samples of myelodysplastic syndrome that had erythroid hyperplasia in bone marrow, acute myelogenous leukemia M1, M2, and chronic myelogenous leukemia. Expression profiles of normal members of the erythroid lineage suggest that TfR2-alpha may be a useful marker of early erythroid precursor cells. The clinical significance of TfR2-alpha expression in leukemia cells remains to be determined.

[Undifferentiated blastic cell crisis of chronic myelogenous leukemia with myeloblastic tumor in the skin].

A 54-year-old female, who had been treated for 4 years in the chronic phase of chronic myelogenous leukemia (CML) was admitted for management of a CML blastic crisis. Blast cells showed strong positive expression of CD7 and HLA-DR, and weakly expressed CD2, CD5 and CD10, as well. The cells were peroxidase negative in peripheral blood and bone marrow. An undifferentiated blastic crisis was diagnosed and she was treated with Interferon-alpha and VP(vincristine 2 mg/week; prednisolone 30 mg/day). A 5-7 mm in diameter tumor in the skin of the anterior right chest appeared one week after VP therapy. The tumor consisted of blasts which were CD13, CD33 and peroxidase positive, unlike the peripheral undifferentiated blasts. This is a rare case of mixed blast crisis with an increase in undifferentiated blasts in peripheral blood and bone marrow, and myeloblastic tumor formation in the skin.

A surgical case of solitary plasmacytoma of rib origin with biclonal gammopathy.

Localized solitary plasmacytoma of the bone (SPB) is a rare disease and is characterized by only one or two isolated bone lesions with no evidence of disease dissemination. A previously healthy 44-year-old male was admitted for evaluation of an abnormal radiographic shadow in the left middle lung field with symptoms of left back pain. Radiological evaluation revealed a peripheral opacity in the left chest wall, which was highly suspected to be a chest wall tumor. CT-guided transcutaneous needle biopsy of the tumor was performed and the specimens showed a monomorphous population of mature plasma cells. The bone marrow biopsy findings revealed no evidence of myeloma and bone scanning revealed only abnormal accumulation in the left seventh rib. He had mild M-proteins in a urine sample and Bence-Jones protein was detected. Immunoelectrophoresis revealed mild biclonal gammopathy of Bence-Jones protein of both the kappa and lambda light-chain types. Under a diagnosis of solitary bone plasmacytoma, preoperative radiation therapy with doses of 40 Gy for the tumor was performed. He underwent complete en bloc resection of the chest wall, including one-third of the left sixth and seventh ribs, the intercostal muscle and the parietal pleura. The protein abnormalities in the urine sample disappeared following surgical resection. Adjuvant chemotherapy using melphalan and prednisolone was performed. He is doing well without evidence of tumor recurrence 2 years following his initial diagnosis.

Transferrin receptor 2-alpha supports cell growth both in iron-chelated cultured cells and in vivo.

In most cells, transferrin receptor (TfR1)-mediated endocytosis is a major pathway for cellular iron uptake. We recently cloned the human transferrin receptor 2 (TfR2) gene, which encodes a second receptor for transferrin (Kawabata, H., Yang, R., Hirama, T., Vuong, P. T., Kawano, S., Gombart, A. F., and Koeffler, H. P. (1999) J. Biol. Chem. 274, 20826-20832). In the present study, the regulation of TfR2 expression and function was investigated. A select Chinese hamster ovary (CHO)-TRVb cell line that does not express either TfR1 or TfR2 was stably transfected with either TfR1 or TfR2-alpha cDNA. TfR2-alpha-expressing cells had considerably lower affinity for holotransferrin when compared with TfR1-expressing CHO cells. Interestingly, in contrast to TfR1, expression of TfR2 mRNA in K562 cells was not up-regulated by desferrioxamine (DFO), a cell membrane-permeable iron chelator. In MG63 cells, expression of TfR2 mRNA was regulated in the cell cycle with the highest expression in late G(1) phase and no expression in G(0)/G(1). DFO reduced cell proliferation and DNA synthesis of CHO-TRVb control cells, whereas it had little effect on TfR2-alpha-expressing CHO cells when measured by clonogenic and cell cycle analysis. In addition, CHO cells that express TfR2-alpha developed into tumors in nude mice whereas CHO control cells did not. In conclusion, TfR2 expression may be regulated by the cell cycle rather than cellular iron status and may support cell growth both in vitro and in vivo.

Cyclin A1 expression in leukemia and normal hematopoietic cells.

Human cyclin A1 is a newly cloned, tissue-specific cyclin that is prominently expressed in normal testis. In this study, we showed that cyclin A1 was highly expressed in a subset of leukemia samples from patients. The highest frequency of cyclin A1 overexpression was observed in acute myelocytic leukemias, especially those that were at the promyelocyte (M3) and myeloblast (M2) stages of development. Cyclin A1 expression was also detected in normal CD34(+) progenitor cells. The expression of cyclin A1 increased when these cells were stimulated to undergo myeloid differentiation in vitro. Taken together, our observations suggest that cyclin A1 may have a role in hematopoiesis. High levels of cyclin A1 expression are especially associated with certain leukemias blocked at the myeloblast and promyelocyte stages of differentiation.

DNA damage induces p21 protein expression by inhibiting ubiquitination in ML-1 cells.

We previously reported that deferoxamine, an iron chelating agent, induced p53 and cell accumulation in the G1 phase of ML-1 cells in the same way as the DNA damaging agent, etoposide. Etoposide treatment increased expression of the p21 gene, a cyclin kinase inhibitor, at both the mRNA and protein levels. However, deferoxamine treatment only increased the p21 mRNA level without the appearance of a detectable protein product. A substrate for cyclin kinase, pRB, was unphosphorylated by etoposide treatment, but remained unaffected by deferoxamine, indicating that p21 was functional after etoposide, but not after deferoxamine treatment. Therefore, in the present study, we investigated the involvement of the ubiquitin proteasome pathway in post-transcriptional regulation of p21. By the addition of lactacystin, a proteasome inhibitor, to deferoxamine treatment, the level of unubiquitinated p21 protein product was similar to that induced by etoposide treatment, and the ubiquitinated p21 bands became apparent. After etoposide treatment, the level of ubiquitinated p21 was diminished and a high level of unubiquitinated p21 expression was observed. We concluded that (1) efficient expression of p21 protein requires inhibition of the ubiquitin-proteasome pathway, and (2) DNA damage inhibits the ubiquitination of p21.

Evaluation by multivariate analysis of the differentiation inhibitory factor nm23 as a prognostic factor in acute myelogenous leukemia and application to other hematologic malignancies.

The differentiation inhibitory factor nm23 can inhibit the differentiation of murine and human myeloid leukemia cells. We recently reported that nm23 genes were overexpressed in acute myelogenous leukemia (AML), and a higher level of nm23-H1 expression was correlated with a poor prognosis in AML, especially in AML-M5 (acute monocytic leukemia). To evaluate the importance of nm23 expression as a prognostic factor in AML, we compared it with other putative prognostic factors in AML. An analysis of the correlation between nm23 expression and the clinical parameters of 110 patients with AML demonstrated that increased nm23-H1 mRNA levels were associated with resistance to initial chemotherapy and with reduced overall survival. Multivariate analysis using Cox's proportional hazard model also showed that elevated nm23-H1 mRNA levels significantly contributed to the prognosis of patients with AML. Especially in AML-M5, nm23-H1 status was the most important prognostic factor. Furthermore, to determine whether we can apply the results observed in AML to other hematologic malignancies, we investigated the relative levels of nm23-H1 and nm23-H2 transcripts in 149 patients with hematologic neoplasms, including 110 with de novo AML, 9 with de novo acute lymphoblastic leukemia, 14 with myelodysplastic syndrome, 16 with chronic myelogenous leukemia (CML), and 5 normal subjects by the reverse transcriptase-polymerase chain reaction. Expression of nm23-H1 was significantly higher in all the hematologic neoplasms, except CML in chronic phase, than in normal blood cells. nm23 may have a prognostic effect in these hematologic malignancies as well as in AML.

C/EBP-epsilon: chromosomal mapping and mutational analysis of the gene in leukemia and preleukemia.

We and others have cloned a novel human gene CCAAT/enhancer-binding protein epsilon (C/EBP-epsilon) encoding a member of the C/EBP gene family. It is exclusively expressed in myeloid and T-lymphoid cells and appears to have an important role in inducing expression of several myeloid-specific genes. We used a polymerase chain reaction (PCR)-based technique to examine DNA from 93 hamster/human radiation hybrid clones in order chromosomally to map C/EBP-epsilon to 14q11.2 (between D14S264 and D14S275) which is telomeric to the T-cell receptor alpha and delta genes and centromeric to several other myeloid gene products including Cathepsin G (CTSG) and Chymase-1 (CMA1). To determine whether C/EBP-epsilon behaves as an altered tumor-suppressor gene, samples from patients with acute myelogenous leukemia (AML) and myelodysplastic syndrome (MDS) evolving to AML were studied for loss of heterozygosity (LOH) using microsatellite sequences that we identified within 0.2 kb of the amino-terminus of the human C/EBP-epsilon gene. Allelic loss of the C/EBP-epsilon gene was detected in four out of 20 (20%) evolving MDS cases and in none of the 17 AML and 17 T-cell leukemia cases. Mutational analysis of the gene was performed using PCR-SSCP on 37 AML and 40 MDS cases including those with LOH at the gene. No abnormalities were found suggesting that the altered gene in this region is not C/EBP-epsilon. Also, C/EBP-epsilon was examined by Southern blot analysis on DNA samples from 20 AML patients and 10 AML cell lines. No rearrangements or amplifications of the gene were detected. Taken together, we have mapped C/EBP-epsilon to 14q11.2, a region containing other myeloid and T-lymphoid specific genes. Furthermore, no structural alterations were detected in the C/EBP-epsilon gene.

A novel, myeloid transcription factor, C/EBP epsilon, is upregulated during granulocytic, but not monocytic, differentiation.

Human C/EBP epsilon is a newly cloned CCAAT/enhancer-binding transcription factor. Initial studies indicated it may be an important regulator of human myelopoiesis. To elucidate the range of expression of C/EBP epsilon, we used reverse transcription-polymerase chain reaction (RT-PCR) analysis and examined its expression in 28 hematopoietic and 14 nonhematopoietic cell lines, 16 fresh myeloid leukemia samples, and normal human hematopoietic stem cells and their mature progeny. Prominent expression of C/EBP epsilon mRNA occurred in the late myeloblastic and promyelocytic cell lines (NB4, HL60, GFD8), the myelomonoblastic cell lines (U937 and THP-1), the early myeloblast cell lines (ML1, KCL22, MDS92), and the T-cell lymphoblastic leukemia cell lines CEM and HSB-2. For the acute promyelocytic leukemia cell line NB4, C/EBP epsilon was the only C/EBP family member that was easily detected by RT-PCR. No C/EBP epsilon mRNA was found in erythroid, megakaryocyte, basophil, B lymphoid, or nonhematopoietic cell lines. Most acute myeloid leukemia samples (11 of 12) from patients expressed C/EBP epsilon. Northern blot and RT-PCR analyses showed that C/EBP epsilon mRNA decreased when the HL60 and KG-1 myeloblast cell lines were induced to differentiate toward macrophages. Similarly, Western blot analysis showed that expression of C/EBP epsilon protein was either unchanged or decreased slightly as the promyelocytic cell line NB4 differentiated down the macrophage-like pathway after treatment with a potent vitamin D3 analog (KH1060). In contrast, C/EBP epsilon protein levels increased dramatically as NB4 cells were induced to differentiate down the granulocytic pathway after exposure to 9-cis retinoic acid. Furthermore, very early, normal hematopoietic stem cells (CD34+/CD38-), purified from humans had very weak expression of C/EBP epsilon mRNA, but levels increased as these cells differentiated towards granulocytes. Likewise, purified granulocytes appeared to express higher levels of C/EBP epsilon mRNA than purified macrophages. Addition of phosphothiolated antisense, but not sense oligonucleotides to C/EBP epsilon, decreased clonal growth of HL-60 and NB4 cells by about 50% compared with control cultures. Taken together, our results indicate that expression of C/EBP epsilon is restricted to hematopoietic tissues, especially myeloid cells as they differentiate towards granulocytes and inhibition of its expression in HL-60 and NB4 myeloblasts and promyelocytes decreased their proliferative capacity. Therefore, this transcriptional factor may play an important role in the process of normal myeloid development.

Molecular analysis of the cyclin-dependent kinase inhibitor genes, p15, p16, p18 and p19 in the myelodysplastic syndromes.

The myelodysplastic syndromes (MDS) are a heterogeneous group of clonal blood disorders characterized by dyshematopoiesis with a frequent evolution to acute leukemia. Chromosomal deletions rather than translocations are the predominant karyotypic abnormalities in MDS, suggesting a recessive mechanism in the pathogenesis of MDS, such as inactivation of tumor suppressor genes. A group of cyclin-dependent kinase inhibitors, p15 (INK4B), p16 (INK4A), p18 (INK4C) and p19 (INK4D), are candidate tumor suppressor genes. To determine whether genetic alterations of these genes play an important role in the development and/or progression of MDS, we examined 46 samples from MDS patients by Southern blotting, single-strand-conformation polymorphism (SSCP) using polymerase chain reaction (PCR) and sequencing of DNA. These samples included 13 refractory anemias (RA), four refractory anemias with ringed sideroblasts (RARS), 16 refractory anemias with an excess of blasts (RAEB), eight refractory anemias with an excess of blasts in transformation (RAEB-T) and five chronic myelomonocytic leukemia (CMMoL) samples. Except for allelic polymorphisms or silent point mutations, no alterations of coding regions of these four CDKI genes were identified. In summary, genetic abnormalities of the p15, p16, p18 and p19 genes are rare events in the development and/or progression of MDS.

Alterations of the cyclin-dependent kinase inhibitor p19 (INK4D) is rare in hematopoietic malignancies.

Cyclin-dependent kinase inhibitors (CDKIs) can be classified into two groups based on the structure of the proteins. One group includes the p21 (CIP1, WAF1, CAP20), p27 (Kip1), and p57 (Kip2) CDKIs, which contain a homologous amino-terminal cyclin-dependent kinase (cdk) inhibitory domain. The p16 (INK4A), p15 (INK4B), and p18 (INK4C) CDKIs, which have an ankyrin repeat motifs, belong to the other group. The p16 and p15 CDKI genes are very frequently altered in a variety of cancers including hematopoietic malignancies. The p19 (INK4D) gene is a newly cloned CDKI which belongs to the latter group. To determine if p19 genetic alterations play a role in hematopoietic malignancies, we examined DNA from 45 childhood newly diagnosed acute lymphocytic leukemias (ALLs), 30 acute myeloblastic leukemias (AMLs), 10 chronic myelocytic leukemias (CMLs), 45 adult T cell leukemias (ATLs), 70 non-Hodgkin's lymphomas (NHLs), and 20 multiple myelomas (MM) as well as 14 ALL, 20 AML, two ATL, and five lymphoma cell lines. Using Southern blot analysis, one homozygous deletion of the p19 gene was detected in a human immunodeficiency virus (HIV)-related Burkitt-like lymphoma sample. No point mutations in any of the samples were found by polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) analysis. Our investigation suggests that alterations of p19 do not play an important role in the development of most hematopoietic malignancies.

[The role of cytokines on hyperleukocytosis associated with acute promyelocytic leukemia induced by all-trans retinoic acid therapy].

The mechanism of the cause of hyperleukocytosis induced by differntiation induction therapy of acute promyelocytic leukemia (APL) by all-trans retinoic acid (ATRA) was studies. Of 11 patients treated by ATRA in our hospital, 3 developed hyperleukocytosis. Moreover, 2 out of 4 patients with retinoic acid syndrome (ATRA syndrome) had hyperleukocytosis. Using the patients' leukemia cells as test material, we obteined the following results. In the presence of ATRA at a concentration that induced differentiation in vitro, promotion or supression of differentiation and lineage determination during the differentiation of APL cells involved factors other than ATRA, such as cytokines. Moreover, APL cells that differentiated into monocytoid cells possessed the capability of producing endogenous cytokines such as TNF alpha, which might be involved in the development of ATRA syndrome. Compared to cells from patients without hyperleukocytosis they had a stronger TNF alpha producing capability and lower sensitivity to TGF beta, showing hyperdifferentiation in response to ATRA. Depending on the case, those with the same sensitivity to ATRA might show different sensitivities to cytokines.