Telomerase is a ribonucleoprotein enzyme that maintains protective structures at the ends of eukaryotic chromosomes. Earlier findings have supported an association between progressive telomere ...shortening in the chronic phase of chronic myelogenous leukemia and the up-regulation of telomerase activity occurring late in the evolution of the disease. We examined the impact of telomerase inhibition by dominant negative-human telomerase reverse transcriptase (DN-hTERT) on the biological features of BCR-ABL-transformed cells.
We introduced vectors encoding DN-hTERT, wild-type (WT)-hTERT, or a control vector expressing only a drug-resistant marker into Philadelphia chromosome-positive K562 cells and OM9;22 cells and assessed the biological effect of telomerase inhibition on cellular immortality.
Ectopic expression of DN-hTERT resulted in complete inhibition of telomerase activity and reduction of telomere length. The entire population of telomerase-inhibited K562 cells exhibited cytoplasmic blebbling and chromatin condensation, features of apoptosis. In contrast, K562 cells expressing WT-hTERT, which differ from the mutants by only two amino acids, exhibited normal morphology. The evidence of apoptosis in the telomerase-inhibited cells was determined by flow cytometric analysis with APO2.7 monoclonal antibody. We also observed enhanced induction of apoptosis by imatinib seen in DN-hTERT-expressing K562 cells, as compared with WT-hTERT-expressing cells.
These results demonstrate that disruption of telomere maintenance limits the cellular life span of leukemia cells and show that the combined use of imatinib and telomere maintenance inhibition may be effective in the treatment of BCR-ABL-positive leukemia.
The authors determined whether the decrease in lymphocytes after surgery is related to apoptosis.
Surgery induces a profound but transient depletion of circulating lymphocytes, However, the mechanism ...underlying this phenomenon is unclear.
Peripheral blood mononuclear cells were obtained from 18 patients before and after elective surgery and studied for morphologic and biochemical markers of apoptosis, DNA fragmentation, and Fas expression.
The DNA staining of peripheral blood mononuclear cells obtained after surgery, which had been cultured for 24 hours in vitro, showed chromatin condensation and fragmentation of cells into collapsed spheres. Moreover, DNA isolated from these peripheral blood mononuclear cells formed a ladder of oligonucleosomal fragments. However, peripheral blood mononuclear cells obtained before surgery showed neither of these changes. The observation that none of these apoptotic cells ingested latex suggested that they were of lymphocytic origin. Fas-positive lymphocytes increased significantly 2 hours after the start of surgery and returned to preoperative levels by postoperative day 7. Anti-Fas antibody augmented apoptosis, whereas ZB4, a Fas antagonist, inhibited apoptosis in lymphocytes after surgery.
These results indicate that circulating lymphocytes in the early perioperative period are susceptible to Fas-mediated apoptosis, which may cause depletion of circulating lymphocytes after surgery.
Recently, the involvement of Epstein‐Barr virus (EBV) in hydroa vacciniforme (HV)‐like eruptions has been suggested. To elucidate the role of EBV in this disease, we isolated EBV‐infected cell clones ...from peripheral blood mononuclear cells (PBMC) and the skin lesions of a patient with HV‐like eruptions; cells isolated from PBMC were designated SNK‐12, and those from the eruption SNK‐11. Both cells expressed CD16, CD56, and HLA‐DR and had germline configurations of the T‐cell receptor and the immunoglobulin genes, indicating that the cell clones were of NK cell lineage. The analysis of EBV terminal repeats indicated that the cells were monoclonal, had identical clonality, and originated from EBV‐positive cells in the PBMC and eruption. Both clones expressed EBNA‐1, but not EBNA‐2. Although LMP‐1 was weakly detected in SNK‐11, no LMP‐1 was detected in SNK‐12. Interestingly, EBV‐infected cells required less IL‐2 for in vitro growth in the later phase of this disease and this appeared to correlate with the expression of LMP‐1, suggesting that the proliferative capacity of the EBV‐positive NK cells increased during the time course of the disease, and LMP‐1 expression might be responsible for that. This is the first report of the isolation of EBV‐infected cells from the skin lesions of HV‐like eruptions and strongly suggests that the HV‐like eruption in the patient was caused by clonal NK cells with latent EBV infection.
The treatment of activated autologous lymphocyte can lead to a potent antitumor effect with destruction of autologous cancer cells, but potential adverse autoimmune effects due to destruction of ...autologous tissue must also be considered. This study was performed to evaluate whether administration of activated autologous lymphocytes induces autoimmune disease. Patients with advanced cancer, who underwent transfer therapy with activated autologous lymphocytes, were eligible for the study. Informed consent was obtained from 22 patients with hepatocelluler carcinoma, ovarian cancer, gastric cancer, etc. The variation in activated lymphocyte phenotypes was CD3+/HLA-DR+ activated T lymphocytes, 23% to 99%; including CD4+ cells, 4% to 65%; CD8+ cells, 10 to 91%; and CD16+/ICD56+ NK cells, 1% to 59%. Of the 22 patients, levels of antinuclear antibody and/or rheumatoid factor were above normal limits during the study in the following 5 patients: 3 patients showed no marked changes, one patient a slight decrease in rheumatoid factor and one patient a slight increase in antinuclear antibody during the course of treatment, respectively. The values for these markers of the other 17 patients varied within normal limits during treatment. Mild transient fever occurred in several patients as an adverse event. There were no other adverse reactions. No clinical symptoms or signs suggestive of autoimmune disease occurred in any patient during or after treatment. These results suggested that long-term administration of activated autologous lymphocytes does not induce autoimmune disease.
Chronic active Epstein–Barr virus infection (CAEBV) is a syndrome that takes diverse clinical courses and is often associated with lymphoproliferative disorders of T / natural killer (NK)‐cell ...lineage. We describe a patient with CAEBV associated with persistent pharyngeal ulcer, and with subsequent nasal T/NK‐cell lymphoma in her neck lymph nodes and nasopharynx. Immunophenotyping of lymphoid cells showed that the lineage of Epstein–Barr virus (EBV)‐positive cells in the patient was of NK‐cell origin. By means of high‐dose recombinant interleukin‐2, we established an EBV‐positive cell line of NK‐cell lineage from her peripheral blood. Southern blot analysis for the number of terminal repeat sequences of EBV detected three NK‐cell clones in the patient’s lymph node. One of these clones was identical to the established cell line but was not observed in the pharyngeal ulcer, while the other two clones were present in the pharyngeal ulcer. These results suggest that the patient had expansion of the three NK‐cell clones, one of which had proliferative capacity in vitro and was involved in the formation of the lymphoma. Moreover, the results suggest that the proliferative capacity of EBV‐positive cells can be variable even in a single patient, and this variability may explain the clinical diversity in CAEBV.
Human herpesvirus-6B (HHV-6B), a causativeagent of exanthem subitum, infects human adult Tcell leukemia (ATL) cell lines. We established a persistentHHV-6B infection in an ATL cell line, TaY, inthe ...presence of 20 units/ml interleukin-2 (IL-2).The HHV-6B infected culture proliferated with aconstant ratio of infected (1%) to the uninfected(99%) cells. When the IL-2 concentration wasreduced to 5 units/ml, the number of infected cellsin the culture increased transiently by 60% in 11days, a new balance of 25% infected cells and 75%uninfected cells was established thereafter. PCRanalysis confirmed a 125-fold increase in theamount of viral genome in the culture, while thetreatment with ganciclovir reduced the proportionof infected cells, indicating that an efficient replicationof virus was induced in the culture. Both ofthese cultures were maintained in the presence of20 or 5 units/ml IL-2 over one year without loss ofinfected cells. Interestingly, we found that culturescontaining the infected cells grew significantlyfaster than the parental uninfected cells atthe same concentration of IL-2. The infected culturecontinued to grow for 7 days even in the absence ofIL-2. Because the infection induces cell cyclearrest, these results indicate that the HHV-6B-infectedATL cells stimulate the growth of theuninfected cells during persistent infection in culture.
Human herpesvirus-6B (HHV-6B), a causative agent of exanthem subitum, infects human adult T cell leukemia (ATL) cell lines. We established a persistent HHV-6B infection in an ATL cell line, TaY, in ...the presence of 20 units/ml interleukin-2 (IL-2). The HHV-6B infected culture proliferated with a constant ratio of infected (1%) to the uninfected (99%) cells. When the IL-2 concentration was reduced to 5 units/ml, the number of infected cells in the culture increased transiently by 60% in 11 days, a new balance of 25% infected cells and 75% uninfected cells was established thereafter. PCR analysis confirmed a 125-fold increase in the amount of viral genome in the culture, while the treatment with ganciclovir reduced the proportion of infected cells, indicating that an efficient replication of virus was induced in the culture. Both of these cultures were maintained in the presence of 20 or 5 units/ml IL-2 over one year without loss of infected cells. Interestingly, we found that cultures containing the infected cells grew significantly faster than the parental uninfected cells at the same concentration of IL-2. The infected culture continued to grow for 7 days even in the absence of IL-2. Because the infection induces cell cycle arrest, these results indicate that the HHV-6B-infected ATL cells stimulate the growth of the uninfected cells during persistent infection in culture.
Studies on nasal T/natural killer (NK)–cell lymphoma have been hampered by its tendency to cause necrosis. Thus, the establishment of cell lines of this neoplasm would seem to be valuable. This study ...attempted to establish cell lines from primary lesions of this tumor, and successfully obtained 2 novel Epstein-Barr virus (EBV)–positive cell lines, SNK-6 and SNT-8, by means of high-dose recombinant interleukin 2. Flow cytometry showed that SNK-6 had an NK-cell phenotype, CD3−CD4−CD8−CD19−CD56+T-cell receptor (TCR) α/β− TCR γ/δ−, whereas SNT-8 was CD3+CD4−CD8−CD19−CD56+TCR α/β− TCR γ/δ+. These were consistent with immunophenotypes of their original tumors, and the cell lines had monoclonal EBV clones identical to ones in their original tumors. Thus, the cell lines developed from cells forming the primary lesions. Genotypic analysis showed that SNK-6 had unrearranged TCR and immunoglobulin heavy-chain genes, supporting the conclusion that SNK-6 was of NK-cell lineage. On the other hand, SNT-8 had rearranged TCR β-, γ-, and δ-chain genes, and together with its phenotype, SNT-8 proved to be a γδ T-cell line. This is the first report of the establishment of cell lines from primary lesions of nasal T/NK cell lymphomas, and the results demonstrated that there are at least 2 lineages, NK- and γδ T-cell, in this neoplasm. Moreover, it has been suggested that nasal T/NK cell lymphomas of these lineages may belong to the same clinicopathologic entity because both types of cases shared common clinical and histopathologic features.
We reported that human esophageal cancer cell lines (ECC) (YES-1, -2, -3, -4, -5, and -6) produced interleukin-6 (IL-6). We, therefore, investigated the growth effects (3Hthymidine uptake assay and ...direct cell count) of IL-6 on these ECC. IL-6 receptor (R) and GP-130 mRNA were detected in all the ECC, using reverse transcriptase-polymerase chain reaction (RT-PCR) assay, and IL-6R was detected in one (YES-3) by immunohistochemical staining. IL-6, anti-IL-6 monoclonal antibody (mAb), or anti-IL-6R mAb caused no reproducible enhancement or suppression of 3Hthymidine uptake by all six ECC. Direct cell count also revealed that the growth enhancement or suppression by IL-6, anti-IL-6 mAb, or anti-IL-6R mAb was relatively small. Particularly, there was no significant sensitivity of YES-3 cells, which definitely produce IL-6 and express IL-6R for IL-6, anti-IL-6 mAb, or anti-IL6R mAb. These results suggest that some esophageal cancers may produce IL-6 and express IL-6R. However, no major interactions between IL-6 and the growth of human esophageal cancer cell lines were detected in this study.
In this study, we describe the cytological and cytogenetic features of six Epstein‐Barr virus (EBV)‐infected natural killer (NK) cell clones. Three cell clones, SNK‐1, ‐3 and ‐6, were derived from ...patients with nasal T/NK‐cell lymphomas; two cell clones, SNK‐5 and ‐10, were isolated from patients with chronic active EBV infection (CAEBV); and the other cell clone, SNK‐11, was from a patient with hydroa vacciniforme (HV)‐like eruptions. An analysis of the number of EBV‐terminal repeats showed that the SNK cell clones had monoclonal EBV genomes identical to the original EBV‐infected cells of the respective patients, and SNK cells had the type II latency of EBV infection, suggesting that not only the cell clones isolated from nasal T/NK‐cell lymphomas but also those isolated from CAEBV and HV‐like eruptions had been transformed by EBV to a certain degree. Cytogenetic analysis detected deletions in chromosome 6q in five out of the six SNK cell clones, while 6q was not deleted in four control cell lines of T‐cell lineage. This suggested that a 6q deletion is a characteristic feature of EBV‐positive NK cells, which proliferated in the diseased individuals. The results showed that EBV‐positive NK cells in malignant and non‐malignant lymphoproliferative diseases shared common cytological and cytogenetic features.
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