Increased LAK and T cell activation in responding renal cell carcinoma patients after low dose cyclophosphamide,IL-2 andα-IFN

Immunologic monitoring of cancer patients treated with IL-2 might help to determine functions of significance for the clinical outcome. Some immune functions in patients with advanced renal cell carcinoma were studied during treatment with low dose cyclophosphamide,α-interferon and IL-2. Cyclophosphamide (500 mg m⁻²) was given day 1, andα-interferon (3×10⁶ u i.m.) and continuous infusion of IL-2 (18×10⁶ u m⁻² day⁻¹) for days 4–9. The cycle interval was 3 weeks. Two to six cycles were given. Eleven patients entered the study. One patient achieved a partial remission, two patients had a minor response and four had a stable disease (‘responding patients’). NK cell activity (K562) increased in all patients while LAK cell activity (against a renal cell carcinoma cell line, A498) was significantly augmented only in responding patients. In the responder group, there was a significant increase in CD3⁺/HLA-DR⁺ T-cells. In parallel, there was a significant decrease in CD45RA⁺ cells as well as in the CD4/CD8 ratio. These data might indicate an expansion of activated T cells with a reduction of cells with a suppressor phenotype in responding patients. The results corroborate the importance of activation of LAK cells and T cells during IL-2 therapy of cancer patients.     

Expression and Characterization of the Recombinant Human FLT-3 Ligand Extracellular Domain in Pichia Pastors

OBJECTIVE The FLT-3 ligand （fms-like tyrosine kinase receptor-3 ligand, FL） is a recently described growth factor affecting early hematopoietic progenitor cells. The FL plays a key role in the growth and differentiation of primitive hematopoietic cells. To yield a high-level of recombinant human FL protein, a recembinant Pichia Pastoris （P. pastoris）strain was constructed. METHODS An artificial expression frame, with the same encoding protein sequence for the FL extracellular domain cDNA, was synthesized by using favored genetic codons of P. pastoris. P. pastoris strain KM71 cells were transformed with the endonuclease Bgl II linearized recombined plasmid, pPIC9K-FL. The plasmid then was linerized in the 5＇AOX1 site and integrated into the yeast KM71 genome. KM71 was transformed with pPIC9K plasmids as a control for the production of recombinant protein. Southern blotting and Northern blotting tests were used to screen the genotype of the recombined strain. Biological activity was demonstrated in vitro with culturing of CD34＋cells. RESULTS The recombinant human FL protein expressed into the yeast culture supertant was identified on the basis of its molecular weight and Western blotting analysis. Numerous bands were observed in the 10-100 kDa molecular mass range. SDS-PAGE showed that the expressed product, a 20 kDa protein, was secreted into the medium in the form of a soluble molecule. Western-blot analyses showed good antigenicity and specificity against polyclonal antibodies. A sharp band and a smeared band were observed at a molecular mass of approximately 20 kDa by Western blotting. The recombinant human FL protein was the major protein component observed in the culture supernatant. The highest yield （108 mg/L） was obtained when expression was induced with 0.5% methanol for 96 h. Deglycosylation with PNGase F resulted in a decrease in apparent molecular mass from 20 kDa to 18kDa forming three bands all of which were also detected by rabbit anti-FL antibodies, Culturing of CD34＋ cells in the presence of KM71pPIC9K-FL over 7 days increased 2.9 fold, while in the control group they increased only 1,5 fold. The biological assay showed that the expressed product could stimulate the proliferation of CD34＋ hematopoietic cells, CONCLUSION We demonstrated that human FL was secreted into the culture supernatant from P. pastoris, and that this yeast strain was a preferred host for recombinant human FL gene expression. This recombinant strain can provide a convenient process for pharmaceutical application.     

荷CEA-rV的DC增强CD3AK对CEA阳性肿瘤特异性杀伤作用的研究

Objective： To survey the special killing activity of CD3AK on anti-CEA-positive tumor enhanced by umbilical cord blood dendritic cell （DC） loaded with CEA recombinant vaccinia virus （CEA-rV）. Methods： Freshly isolated umbilical blood mononuclear calls （UBMC） were cultivated for 3 h. Suspension cells and attached calls were used to induce CD3AK calls and DC separately. DC was loaded with CEA-rV on the 3rd day to prepare CEA-rV＋DC. CD3AK cells were co-cultured with CEA- rV＋DC on the 8th day, to prepare CEA-rV＋DC＋CD3AK. The killing activity of each effector＇s cell, which included UBMC, CD3AK, DC＋CD3AK and CEA-rV＋DC＋CD3AK, was measured respectively by MTT reduction assay. Results： （1） 4 target cells were con- firmed by CEA monoclonal antibody of rabbit anti-human. Lovo and A549 were really CEA positive cell lines, while Bel-7402 and K562 were CEA negative cell lines. （2） It was showed by flow-cytometry that the mature DC cultured at 10th day expressed MHC I, II molecules such as CD86, CDS0, CD83 and CD40 highly, but CD123 lowly. The expression rates of CD86, CDS0, CD83 and CD40 was 82.7%, 51.1%, 57.5% and 69.4%, respectively. The appearances and intra-cellular structures of DC were observed through light and electron microscope. The diameter of mature DC was 15-20 μm presented the irregular morphologic appearanca, much prominences and pseudopodium. There were abundant mitochondria and endoplasmic reticulum in DC endochylema. （3） The rates of CD3, CD4, CD8 and CD28 in CD3AK cells group were 2 folds higher than that in UBMC group by FACS. It was said that the numbers of the mature T lymphocyte in CD3AK cells group were much greater than that in UBMC group. （4） The killing activities to 4 target cells of 3 effector＇s cells, which included CEA-rV＋DC＋CD3AK, DC＋CD3AK and CD3AK, were much greater than that of UBMC （P〈0.01）. Moreover, comparing with the killing activities of 4 effector＇s： CEA-rV＋DC＋CD3AK group 〉 DC＋CD3AK group 〉 CD3AK group 〉 UBMC group. It showed that, cytokine, DC and CEA-rV could efficiently elevate the killing activity of UBMC on broad-spectrum tumor cells. （5） Comparing with the killing activities of CEA-rV＋DC＋CD3AK and CD3AK cells to CEA positive and negative cells, the killing activities of CEA-rV＋DC＋CD3AK to CEA positive tumor calls, Lovo and A549 calls （P〈0.01） were remarkably better than that to CEA negative tumor cells BEL-7402 and K562 cells （P〈0.05）. It was said that the CEA-rV＋DC could obviously enhance the killing activity of CD3AK on CEA positive tumor cells. Comparing with the killing activities of CEA-rV＋DC＋CD3AK and DC＋CD3AK cells, the killing activity of CD3AK on CEA negative tumor cells was no statistical difference （P〉0.05）. However, the killing activity to CEA positive cells of CEA-rV＋DC＋CD3AK group was notably higher than that of DC＋CD3AK group. Namely, CEA-rV could distinctly promote the special killing activity to CEA positive tumor cells of CD3AK, but could not do it to CEA negative tumor cells. Conclusion： CEA-rV＋DC could obviously enhance the special killing activity of CD3AK on CEA positive tumor cell lines, while the DC only couldn＇t. The results indicated that the CEA-rV played an important role during the special killing activity of CD3AK cells to CEA positive tumor cells.     

Stimulation of staphylococcal enterotoxin A combined with PML-RARα peptide on the specifical T-cells against NB4 cell line

Objective  To investigate the effects of staphylococcal enterotoxin A (SEA) on the cytotoxicity of T cells stimulated by PML-RARα peptide in vitro. Methods  Peripheral blood mononuclear cells (MNC) from healthy donor were obtained by density gradient centrifugation on Ficoll-Hypaque, MNC were cultured with PML-RARα peptide and SEA for 20 days. After induction, the cytotoxicity of T cells induced against NB4 and K562 cell lines were examined by Cell Counting Kit-8 (CCK-8). The CD4 and CD8 surface markers on the harvested CD3⁺ T cells were detected by flow cytometry (FCM). Results  The cytotoxicity of T cells induced by PML-RARα peptide with SEA was higher than that of T cells induced only by PML-RARα peptide against NB4 cells. The FCM assay showed that the ratio of CD4⁺/CD8⁺ T cells were gradually decreased in both groups of PML-RARα peptide whether with SEA or not at the intervals of day 5,10 and 20 day after induction, but the most significantly decreased by PML-RARα peptide with SEA. Conclusion  The specific cytotoxicity of T cells induced by PML-RARα peptide against NB4 cells could be enhanced with superantigen SEA. Supported by grants from the Science and Technology Commission of Guangdong Province (No. 06025169, No. 2005B50301016), and the Key Laboratory of Pathophysiology of Jinan University.     

The variation of CD4+CD25+ regulatory T cells in the periphery blood and tumor microenvironment of non-small cell lung cancer patients and the downregulation effects induced by CpG ODN

The aim of the study was to observe the variation of CD4⁺CD25⁺ regulatory T cells in periphery blood and tumor microenvironment of non-small cell lung cancer (NSCLC) patients and the effects of CpG ODN. The proportion of CD4⁺CD25⁺ regulatory T cells, Foxp3 gene expression, levels of tumor growth factor-β (TGF-β) and immunoreactive fibronectin-γ (IFN-γ) in the periphery blood of 30 NSCLC patients and 30 healthy volunteers were compared. These indicators were compared before and after CpG ODN treatment. Foxp3 gene expression in the tumor microenvironment of NSCLC patients was also observed. The results showed CD4⁺CD25⁺ regulatory T cell proportion, Foxp3 expression and TGF-β levels in the periphery blood of NSCLC patients were higher than those of healthy volunteers (p < 0.05), and these indicators of patients were significantly decreased after CpG ODN 2006 treatment (p < 0.05). Foxp3 expression in the metastatic lymph nodes was higher than that in the non-metastatic ones of NSCLC patients (p = 0.000). In conclusion, a rise in the proportion of CD4⁺CD25⁺Foxp3⁺ regulatory T cells was demonstrated in the periphery blood and tumor microenvironments of NSCLC patients. CpG ODN 2006 downregulated the CD4⁺CD25⁺Foxp3⁺ regulatory T cells proportion and TGF-β levels in the periphery blood of these patients.     

Bone marrow mesenchymal stem cells from leukemia patients inhibit growth and apoptosis in serum-deprived K562 cells

Background

The regulation of growth and apoptosis in K562 cells by human bone marrow mesenchymal stem cells (MSCs) from leukemia patients was investigated.

Methods

K562 cells were cocultured with leukemic MSCs under serum deprivation. Cell Counting Kit-8 (CCK-8), PI staining, Annexin V/PI binding and FACS assays were used to investigate cell proliferation, cell cycle status, and apoptosis of K562 cells cultures in the presence or absence of 10% serum. Western blotting was used to determine the levels of Akt, phosphorylated Akt (p-Akt), the BCL-2 family member Bad, and phosphorylated Bad (p-Bad) proteins in K562 cells after coculturing with MSCs. The effects of {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 (a specific inhibitor of PI3K) on protein expression were also determined.

Results

K562 cell proliferation was inhibited by coculture with MSCs and the dominant cell cycle was the G₀-G₁ phase. The proportion of apoptotic K562 cells was decreased and the levels of p-Akt and p-Bad were upregulated after exposing K562 cells to MSCs. However, when {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 was used, p-Akt and p-Bad proteins inK562 cells showed a significant reduction, while no distinct variation was seen in the nonphosphorylated Akt and Bad protein levels.

Conclusion

Leukemic MSCs can inhibit K562 cell expansion and modulate the cell cycle to a state of relative quiescence. This allows the K562 cells to endure adverse conditions such as serum starvation. The PI3K-Akt-Bad signaling pathway may be involved in this antiapoptotic process via phosphorylation of the Akt and Bad proteins. Blocking MSC-induced transduction of the PI3K-Akt-Bad pathway may be a potential strategy for a targeted therapy to combat leukemia.     

CD80 expression in an HLA-A2-positive human non-small cell lung cancer cell line enhances tumor-specific cytotoxicity of HLA-A2-positive T cells derived from a normal donor and a patient with non-small cell lung cancer

To generate non-small cell lung cancer (NSCLC)–reactive lymphocytes, we transfected an HLA-A2-expressing human NSCLC line (1355) with the cDNA encoding the lymphocyte co-stimulatory molecule CD80. Following selection in G418, 1355.7 demonstrated stable cell-surface expression of CD80. Allogeneic mixed lymphocyte tumor cell cultures (MLTCs) were established in 600 IU/ml IL-2 using HLA-A2⁺ normal donor peripheral blood mononuclear cells (PBMCs) stimulated with 1355-P (parental), 1355.7 or IL-2 alone. In 7 of 9 MLTCs, those stimulated with 1355.7 demonstrated enhanced growth after 30 to 45 days of culture. The predominant lymphocyte to grow in all MLTCs was a CD3⁺αβ⁺CD4⁺ T cell. In one case, lymphocytes stimulated with 1355.7 (MLTC 2389.7) exhibited preferential lysis of 1355. MLTC 2389.7 was cloned by limiting dilution, and 2 resultant cloids were shown to be NSCLC-reactive and dependent on both MHC class I and CD3 in their recognition of tumor cells. Additionally, allogeneic MLTCs were established using three HLA-A2⁺ NSCLC patients' PBMC. The predominant lymphocyte to grow in these MLTCs was a CD3⁺ αβ⁺CD8⁺ T cell. In cytotoxicity studies, MLTC-UKY25.7 demonstrated preferential lysis of 1355-P, 1355.7 and an HLA-A2⁺ NSCLC cell line, 1650. Lymphocytes from this MLTC did not lyse K562, Daudi or an HLA-A2^– NSCLC cell line, 647. Our data suggest that CD80-expressing NSCLC tumor cells may enhance the generation of specific CTLs in vitro. These CTLs could be important reagents for use in cellular immunotherapy and/or in isolating tumor antigens for potential tumor vaccine development. Int. J. Cancer 78:685–694, 1998. © 1998 Wiley-Liss, Inc.     

Trichosanthin down-regulated p210Bcr-Abl and enhanced imatinib-induced growth arrest in chronic myelogenous leukemia cell line K562

Purpose Trichosanthin (TCS), an active component extracted from the root tubers of traditional Chinese medical herb Tian-Hua-Fen of the Cucurbitaceae family, has long been used for medical purpose in China; there is increasing interest in developing TCS as cancer therapeutic agents. The present study was to investigate the growth arrest of K562 cells and its molecular mechanisms, which the drugs induced by TCS and the possible functional interaction of TCS with imatinib (STI571) to K562 cells. Methods Trypan blue exclusive staining was used to access the cell growth inhibition; western blot was used to evaluate the p210^Bcr-Abl, phosphorylated tyrosine kinase (PTK), and some signaling molecules involving in cell proliferation and apoptosis in K562 cells. Results TCS and imatinib inhibited K562 cells at a time- and dose-dependent manners, respectively; TCS down-regulated p210^Bcr-Abl at a time- and dose-dependent manners; TCS synergistically enhanced imatinib-induced K562 cell growth arrest and down-regulation of p210^Bcr-Abl, PTK activities, procaspase-3, Hsp90,NF-κB and PKC. Conclusion The results suggest that TCS not only by itself involves but also synergizes activities of imatinib to induce K562 cell growth arrest, down-regulation of p210^Bcr-Abl and its downstream signals and to stimulate the effect of the tyrosine kinase inhibition.     

T Cell Density and Location Can Influence the Prognosis of Ovarian Cancer

The aims of this study were to examine the significance of CD3+ cells in patients with epithelial ovarian cancer and to determine their influence on the disease in relation to their location within tumours. A 157-core tissue-microarray constructed from primary ovarian cancer patients treated at Nottingham-University-Hospitals (2000–2007) was stained for the T-cell marker CD3. The number of CD3+ cells in direct contact with tumour cells was counted per tumour area. These were considered as “intra-tumoural T-cells (ITTC)”. Cores were divided into CD3 ‘high’ or ‘low’ density tumours. “Stromal T-cells (STC)” were assigned as ‘positive’ or ‘negative’. The study population had a median follow-up time of 36-months (0–75). The number of ITTC counted in tumour cores ranged between 0 and 184/mm². 90-tumours-(57%) were found to be in the “low-density” rubric, while 56-(36%) were of a “high-density” T-cell population. STC were found in 118-cores-(75%)-compared to 22-cores-(14%)-negative cores. Higher number of ITTC correlated with lower-grade-(p = 0.045), tumour-type-(p = 0.034), and longer-median-survival-times (57-versus 37-months for high-and low-ITTC densities, respectively, p = 0.038). This relationship was reversed when tumours were infiltrated by CD3+ cells in the stroma, predicting worse-survival (Log-rank-test, p = 0.028). Combining ITTC with STC produced an interesting pattern where the ITTC-low/STC + ve had the worst prognosis (p = 0.003). Infiltration of ovarian cancer by T-cells can influence its prognosis depending on the location of these cells (intra-tumoural-versus-stromal). The former predicts improved survival, while the latter is probably contributing to tumour progression and, in turn, worse survival.     

Melanoma‐targeted chemo‐thermo‐immuno (CTI)‐therapy using N‐propionyl‐4‐S‐cysteaminylphenol‐magnetite nanoparticles elicits CTL response via heat shock protein‐peptide complex release

Melanogenesis substrate, N‐propionyl‐4‐S‐cysteaminylphenol (NPrCAP) is specifically taken up by melanoma cells and inhibits their growth by producing cytotxic free radicals. By taking advantage of this unique chemical agent, we have established melanoma‐targeting intracellular hyperthermia by conjugating NPrCAP with magnetite nanoparticles (NPrCAP/M) upon exposure to an alternating magnetic field (AMF). This treatment causes cytotoxic reaction as well as heat shock responses, leading to elicitation of antitumor immune response, which was proved by tumor rechallenge test and CTL induction. We found the level of heat shock protein 72 (Hsp72) to be increased in the cell lysate and culture supernatant after intracellular hyperthermia. Melanoma‐specific CD8⁺ T‐cell response to dendritic cells loaded with hyperthermia‐treated tumor lysate was enhanced when compared with non‐treated tumor lysate. When heat shock protein, particularly Hsp72, was immuno‐depleted from hyperthermia‐treated tumor cell lysate, specific CD8⁺ T‐cell response was abolished. Thus, it is suggested that antitumor immune response induced by hyperthermia using NPrCAP/M is derived from the release of HSP‐peptide complex from degraded tumor cells. Therefore, this chemo‐thermo‐immuno (CTI)‐therapy might be effective not only for primary melanoma but also for distant metastasis because of induction of systemic antimelanoma immune responses. (Cancer Sci 2010)     

Intratumoral T cell subset ratios and Fas ligand expression on brain tumor endothelium

Summary Introduction: T cell presence in TIL, and the ratio of CD8⁺ and CD4⁺ T cell subsets in particular, can correlate with tumor prognosis in some tumors, although the significance of such infiltration into glioma is controversial. However, gliomas represent a lower extreme in their extent of T cell infiltration, and are thus useful in assessing factors that can decrease T cell presence within tumor tissue. Fas ligand, a pro-apoptotic cell surface protein, may play a key role in reduction of T cells in tumor tissue. Objective: To assess the level of FasL expression on brain tumor endothelium and to correlate this with relative levels of CD4⁺ and CD8⁺ T cell subsets in TIL from brain tumors. Methods: CD3⁺, CD4⁺, and CD8⁺ cells were quantified in fresh TIL by flow cytometry. Paraffin embedded sections of tumors, including meningiomas and gliomas as well as extracranial malignancies, underwent immunohistochemical staining for FasL and Von-Willebrand’s factor (Factor VIII) to determine expression levels of endothelial FasL. Results: FasL expression was high in aggressive intracranial malignancies compared to more indolent neoplasms, and correlated inversely with CD8⁺/CD4⁺ TIL ratios in all tumor classes combined (ANOVA,p<0.05). Conclusion: Low levels of T cells within TIL, as well as low CD8⁺/CD4⁺ TIL ratios appear to be a property of parenchymal tumor presence. Together with the inverse correlation seen between FasL expression and CD8⁺/CD4⁺ TIL ratios, the high levels of endothelial FasL expression in gliomas suggests that FasL decreases T cell presence in brain tumors in a subset-selective manner, thus contributing to glioma immune privilege.     

CD3AK/iNOS细胞对白血病细胞体外杀伤的研究

背景与目的:LAK细胞已用于临床移植物净化。CD3AK细胞是CD3单克隆抗体激活的杀伤细胞。本研究旨在经逆转录病毒介导iNOS基因转染人免疫活性细胞CD3AK建立CD3AK/iNOS细胞,并探讨其对白血病细胞株K562及其耐药株K562/ADM的杀伤作用。方法:培养PA317/pLNC-iNOS细胞获得病毒上清;CD3McAb联合小剂量的IL-2激活分离的外周血单个核细胞;含逆转录病毒颗粒的细胞培养上清感染靶细胞CD3AK细胞。测定CD3AK/iNOS、CD3AK细胞培养上清NO含量以及iNOS活性。MTT法观察CD3AK/iNOS、CD3AK对K562和K562/ADM细胞杀伤活性。结果:(1)CD3AK/iNOS、CD3AK中NO含量分别为(378.60±41.57)μmol/L,(98.07±22.31)μmol/L(P<0.001);CD3AK/iNOS、CD3AK中iNOS活性分别为(20.77±2.49)U/ml,(9.81±1.96)U/ml(P<0.001)。(2)MTT法观察CD3AK/iNOS对K562和K562/ADM杀伤活性分别为(64.85±18.13)%,(63.80±9.93)%。CD3AK杀伤活性分别为(45.66±17.46)%,(47.85±12.01)%。CD3AK/iNOS与CD3AK相比差异有显著性(P<0.05)。结论:CD3AK/iNOS分泌的NO含量、iNOS活性较CD3AK明显提高,且对K562及K562/ADM杀伤作用也更强;但CD3AK/iNOS对K562及K562/ADM杀伤作用无显著性差异。     

A CD16/CD30 bispecific monoclonal antibody induces lysis of hodgkin's cells by unstimulated natural killer cells In AND In vivo

In order to target NK cells against the Hodgkin's-derived cell line L540, we developed bispecific monoclonal antibodies (Bi-MAbs) by somatic hybridization of the 2 mouse hybridoma cell lines HRS-3 and A9 which produce monoclonal antibodies (MAbs) with reactivity against the Hodgkin and Reed-Sternberg cell-associated CD30 antigen and the CD 16 antigen (FcγIII receptor), respectively. The CD 16 MAb-producing cell line A9 was selected as a partner for HRS-3 because of its efficiency in inducing lysis of the A9 hybridoma cells by resting NK cells. The hybrid hybridoma cell line HRS-3/A9 produced the supernatant with the strongest bispecific reactivity and was repeatedly subcloned and used for ascites production. Crude supernatant and purified HRS-3/A9 Bi-MAb triggered specific lysis of the CD30⁺ Hodgkin's-derived cell line L540, but not of the CD30^? cell line HPB-ALL by unstimulated peripheral-blood lymphocytes and NK-cell-enriched populations. Moreover, treatment of SCID mice bearing heterotransplanted human Hodgkin's tumors with HRS-3/A9 and human peripheral blood lymphocytes induced specific complete tumor regression in 10/10 animals. We thus report successful tumor treatment in an in vivo model using NK-cell-associated Bi-MAbs and show that the Bi-MAb HRS-3/A9 is an efficient promoter of the anti-tumor effects of NK cells in vitro and in vivo.     

BCG (bacille Calmette-Guérin)-activated lymphocytes (BAK cells) induce apoptosis in urinary bladder carcinoma cell line T24 in vitro

Background. We previously reported the basic characteristics of BCG (bacille Calmette-Guérin)-activated killer (BAK) cells, which exhibited antitumor effects against the bladder cancer cell line T24. Our study suggested that both BCG and BAK cells were responsible for the inhibition of tumor cell proliferation; however, the basic mechanism of BCG or BAK cells in this inhibition was not clear. We here report the antitumor effects of BAK cells, which correlated with the induction of apoptosis in T24 cells. Methods. Lymphocytes were cultured with BCG to examine ³H-thymidine uptake, and the subpopulation was evaluated by immunocytometry. T24 cells were then cultured with BAK cells for the analysis of ³H-thymidine uptake and apoptosis induction by DNA electrophoresis; pathology study, and cell-cycle analysis were also done. Culture supernatants of BAK and T24 cells were also investigated to detect interferon-γ (IFN-γ), interleukin-6 (IL-6), and tumor necrosis factor-α (TNF-α). Results. The ³H-thymidine uptake study of lymphocytes showed that BCG activated the lymphocytes. Evaluation by immunocytometry revealed that CD4⁺ and CD8⁺ T cells were induced by BCG. The ³H-thymidine uptake study of T24 cells revealed that BAK cells inhibited tumor cell proliferation. DNA electrophoresis, the morphological study, and cell-cycle analysis by immunocytometry demonstrated that apoptosis in T24 cells was induced when they were cultured with BAK cells. IFN-γ, IL-6, and TNF-α were detected in the culture supernatants of BAK and T24 cells. Conclusions. Cytokine production and the induction of apoptosis may, together, be the major mechanisms of the antitumor action seen when BAK cells were employed against T24 cells; BAK cells could be employed as clinical effectors against bladder cancer. Received: January 7, 2000 / Accepted: April 27, 2000     

Antitumor activity of daphnane-type diterpene gnidimacrin isolated from Stellera chamaejasme L

The daphnane-type diterpene gnidimacrin, isolated from the root of the Chinese plant, Stellera chamaejasme L., was found to strongly inhibit cell growth of human leukemias, stomach cancers and non-small cell lung cancers in vitro at concentrations of 10⁻⁹ to 10⁻¹⁰ M. On the other hand, even at 10⁻⁸ to 10⁻⁵ M, the small cell lung cancer cell line H69 and the hepatoma cell line HLE were refractory to gnidimacrin. The agent showed significant antitumor activity against murine leukemias and solid tumors in an in vivo system. In K562, a sensitive human leukemia cell line, gnidimacrin induced blebbing of the cell surface, which was completely inhibited by staurosporine at concentrations above 10⁻⁸ M, and arrested the cell cycle transiently to G₂ and finally the G₁ phase at growth-inhibitory concentrations. It inhibited phorbol-12,13-dibutyrate (PDBu) binding to K562 cells and directly stimulated protein kinase C (PKC) activity in the cells in a dose-dependent manner (3–100 nM). Although activation of PKC isolated from refractory H69 cells was observed only with 100 nM gnidimacrin, the degree of activation was lower than that produced by 3 nM in K562 cells. Our results suggest that gnidimacrin acts as a PKC activator for tumor cells and that this mechanism may be responsible for its antitumor activity. © 11996 Wiley-Liss, Inc.     

Expression of caspase-3 in cord blood CD34<Superscript>+</Superscript> cells during culture in vitro

Objective: To investigate the expression and significance of caspase-3 protein in CD34⁺ cells from cord blood (CB) during culturein vitro with different growth factors.Methods: RT-PCR, Western blot and flow cytometry techniques were used to detect the expression of caspase-3 in CD34⁺ CB cells during culturein vitro. Results: Caspase-3 mRNA was constitutively expressed at a low level in freshly isolated CD34⁺ cells. The expression of caspase-3 mRNA and protein was upregulated when these cells were first expanded in suspension culture with growth factors for 3 days. However, only the 32 kDa inactive caspase-3 proenzyme was detected in the freshly isolated CD34⁺ cells as well as during the first 3 days expansion with cytokines. With longer culture timein vitro, especially in the presence of the combination of IL-3, IL-6 and GM-CSF, caspase-3 was activated and a cleavage product of 20 kDa became detectable.Conclusion: Caspase-3 is involved in apoptosis of primitive CB CD34⁺ cells during expansionin vitro. The study was supported by a grant from the National Natural Science Foundation of China (No. 39928010)     

The research on the immuno-modulatory defect of Mesenchymal Stem Cell from Chronic Myeloid Leukemia patients

Overwhelming evidence from leukemia research has shown that the clonal population of neoplastic cells exhibits marked heterogeneity with respect to proliferation and differentiation. There are rare stem cells within the leukemic population that possess extensive proliferation and self-renewal capacity not found in the majority of the leukemic cells. These leukemic stem cells are necessary and sufficient to maintain the leukemia. While the hematopoietic stem cell (HSC) origin of CML was first suggested over 30 years ago, recently CML-initiating cells beyond HSCs are also being investigated. We have previously isolated fetal liver kinase-1-positive (Flk1⁺) cells carrying the BCR/ABL fusion gene from the bone marrow of Philadelphia chromosome-positive (Ph⁺) patients with hemangioblast property. Here, we showed that CML patient-derived Flk1⁺CD31^-CD34^-MSCs had normal morphology, phenotype and karyotype but appeared impaired in immuno-modulatory function. The capacity of patient Flk1⁺CD31^-CD34^- MSCs to inhibit T lymphocyte activation and proliferation was impaired in vitro. CML patient-derived MSCs have impaired immuno-modulatory functions, suggesting that the dysregulation of hematopoiesis and immune response may originate from MSCs rather than HSCs. MSCs might be a potential target for developing efficacious cures for CML.     

Mechanisms of growth stimulation by suramin in non-small-cell lung cancer cell lines

Purpose: Suramin, a polysulfonated naphthylurea, has been shown to be effective in the treatment of several cancers. We have reported that suramin, at dose concentrations higher than 140 μM, exerts growth-stimulatory effects in several non-small-cell lung cancer (NSCLC) cell lines. The purpose of this study was to examine the mechanisms by which suramin exerts this growth-stimulatory effect in NSCLC cells. Methods: NCI-H596 cells were treated with agarose-immobilized suramin, directly or by addition on cell culture inserts, after which growth was determined by [³H]thymidine incorporation. PPADS, a specific purinergic receptor antagonist, was used to determine whether suramin acts via purinergic receptors. The effect of suramin on epidermal growth factor receptor (EGFR) was determined by analyzing receptor phosphorylation and dimerization. XAMR 0721, a suramin analogue containing only one of the two polysulfonated arms, was also analyzed for its effects on growth and EGFR activation. Results: Agarose-immobilized suramin stimulated NCI-H596 cell growth, but only when added directly to the cells. When the suramin-conjugated beads were added to the cells on cell culture inserts, which preclude an interaction with the cell surface but allow interaction with the culture medium, there was no effect on proliferation. PPADS had no effect on the growth stimulation by suramin; however suramin treatment resulted in rapid phosphorylation and dimerization of EGFR. Treatment with XAMR 0721 did not affect growth or tyrosine phosphorylation and dimerization of EGFR. Conclusions: Suramin need not enter NCI-H596 cells to exert its growth-stimulatory effect, nor is this effect mediated by an interaction with soluble growth factors. Rather, it appears that suramin acts via an interaction with EGFR, but not with purinergic receptors. Received: 4 May 1998 / Accepted: 14 August 1998     

Natural killer cell activity in chronic lymphocytic leukemia patients treated with fludarabine

 Fludarabine, the 5′-monophosphate of 9-β-D-arabinofuranosyl-2-fluoroadenine (FaraAMP), is effective in the treatment of chronic lymphocytic leukemia (CLL) and has been demonstrated to increase natural killer (NK) cell lytic activity (NK_a) in humans and mice. To determine the effect of FaraAMP on NK cells in CLL, we analyzed NK_a toward K562 targets after in vitro incubation with FaraAMP and after in vivo exposure to fludarabine. Pretreatment analysis of peripheral blood from 12 CLL patients (9 untreated) revealed: median number of NK cells 500/μl (range 290–1160); median NK_a lytic unit₃₀/10⁶ cells (range 5–80). These results were similar to those from healthy adult donors. After exposure to 3, 30, or 300 μM FaraAMP, the median maximum stimulation index (NK_a FaraAMP/NK_a) was 1.2 (range 0.9–1.5), within the range observed in normal adults. FaraA also stimulated NK_a in vitro toward autologous CLL cells in two of five patients as measured by a dye-exclusion assay. In three patients following three or more treatment courses of fludarabine (30 mg/m² per day for 5 days) the NK cell number and NK_a were maintained near pretreatment values. Phenotypic analysis of the peripheral mononuclear cells in 34 consecutive CLL patients revealed a marked reduction in CD5/CD20 and CD4 cell numbers after three courses of fludarabine with less effect on CD8 and CD56 cells. These results indicate that fludarabine spares NK cells and may stimulate NK_a in some CLL patients. Received: 22 December1994/Accepted: 14 May 1995