Biochemical changes associated with the development of resistance to 5-azacytidine in AKR leukemic mice

Resistance to 5-azacytidine, a potent inhibitor of the growth of mouse lympaoid leukemia cells in vivo, was achieved in an inbred strain of AKR mice in the third transplant period. The incorporation of orotic acid into the livers of such resistant mice was more than six times lower than in animals carrying the 5-azacytidine-sensitive parent strain of leukemia. The incorporation of orotic acid by 5-azacytidine-sensitive mice was inhibited by 5-azacytidine twice as much as in 5-azacytidine-resistant animals. The incorporation of uridine, cytidine, thymidine, deoxycytidine, and of guanosine into nucleic acids was also significantly depressed in 5-azacytidine-resistant leukemic cells. Uridine kinase activity was decreased by 20%, while uridine phosphorylase activity was diminished by 31–50%. The resistant leukemic mice were cross-resistant to 5-fluorouracil; in contrast, however, their sensitivity to aminopterin was increased significantly.     

Induction of apoptosis by dietary polyunsaturated fatty acids in human leukemic cells is not associated with DNA fragmentation

Apoptosis is important in anticancer strategy. In this study, bivariate annexin V/PI flow cytometry showed that dietary polyunsaturated fatty acids, arachidonic acid (AA) and eicosapentaenoic acid (EPA), induced apoptosis in human leukemic HL-60 but not K-562 cells. Results from DNA-PI flow cytometry and TUNEL flow cytometry illustrated that neither AA nor EPA induced DNA fragmentation in the leukemic cells. These findings suggested that the AA- and EPA-induced apoptosis might not associate with endonucleases activation, and DNA fragmentation could not be used as a sole criterion to identify apoptotic cells.     

Generation of cytotoxic donor CD8+ T cells against relapsing leukemic cells following allogeneic transplantation by stimulation with leukemic cell- or leukemic lysate pulsed donor cell-derived dendritic cells

To treat leukemia relapse after allogeneic hematopoietic stem cell transplantation (HSCT), we investigated the possibility of immunotherapy using donor CD8+ T cells that were generated by stimulating leukemic cell-derived dendritic cells (leukemic-DCs) or leukemic cell lysate pulsed donor cell-derived DCs (donor-DCs). Leukemic- and donor-DCs were generated from mononuclear cells of patients and CD14+ cells of HLA-matched donors, respectively. The expression of CD80, CD83, CD86, CD1a, and CD40 on leukemic-DCs was significantly lower than that on donor-DCs. Donor-DCs exhibited a higher capacity to stimulate allogeneic T cells compared with leukemic-DCs. Donor CD8+ T cells stimulated by leukemic- or donor-DCs were more cytotoxic than unprimed CD8+ T cells, and slightly higher cytotoxicity was observed with donor-DCs compared to leukemic-DCs. This study indicates that leukemic- or donor-DCs pulsed with leukemic cell lysates can effectively prime donor cytotoxic T cells in vitro, and that they may be used as a potential alternative tool for treating leukemic patients who relapse after allogeneic HSCT.     

Regulation of the cyclin A1 protein is associated with its differential subcellular localization in hematopoietic and leukemic cells

An important role of the cell cycle regulatory protein cyclin A1 in the development of acute myeloid leukemia (AML) was previously demonstrated in a transgenic mouse model. We have now turned our attention to study specific aspects of the activity and subcellular distribution of cyclin A1 using bone marrow samples from normal donors and patients with AML, as well as leukemic cell lines. We show that the localization of cyclin A1 in normal hematopoietic cells is nuclear, whereas in leukemic cells from AML patients and cell lines, it is predominantly cytoplasmic. In leukemic cell lines treated with all-trans retinoic acid (ATRA), cyclin A1 localized to the nucleus. Further, there was a direct interaction between cyclin A1 and cyclin-dependent kinase 1, as well as a major ATRA receptor, RARalpha, in ATRA-treated cells but not in untreated leukemic cells. Our results indicate that the altered intracellular distribution of cyclin A1 in leukemic cells correlates with the status of the leukemic phenotype.     

Resistance of acute myeloid leukemic cells to the triterpenoid CDDO-Imidazolide is associated with low caspase-8 and FADD levels

The synthetic triterpenoid CDDO-Im-induced apoptosis of patient-derived AML blasts: 11/25 AMLs were highly sensitive, while the remaining were moderately sensitive to CDDO-Im. The addition of TRAIL significantly potentiated the cytotoxic effect of CDDO-Im, through mechanisms involving the induction of TRAIL-R1/TRAIL-R2 and downmodulation of TRAIL-R3/TRAIL-R4. Biochemical studies showed that CDDO-Im: induced a rapid and marked GSH depletion and antioxidants (GSH or NAC) completely inhibited its pro-apoptotic effect; sequentially activated caspase-8, -9 and -3; caspase inhibitors partially protected AML blasts from CDDO-Im-induced apoptosis; resistance of AML blasts to CDDO-Im-induced apoptosis correlated with low caspase-8/FADD and high Bcl-X_L expression in leukemic blasts.     

Enhanced AKR leukemogenesis by the dual tropic viruses. I. The time and site of origin of potential leukemic cells

The occurrence of potential leukemia cells (PLC) among bone marrow, spleen, and thymus of AKR mice during the preleukemic period was tested by an in vivo transplantation bioassay. The presence of PLC in 30- and 75-day-old AKR mice was demonstrated mostly among bone marrow cells, less in spleen, and was lacking in thymus. Occurrence of PLC in young AKR mice was shown to be thymus independent. However, progression of PLC from young donors (14-80 days old) into overt leukemia following transplantation into F1 recipients was shown to be dependent on specific host conditions including an intact thymus and an Fv-1nn allele. In contrast, PLC from 7-9-month-old AKR mice or frank leukemic cells when transplanted grew in any intact or thymectomized histocompatible host, thereby indicating their autonomous growth state. Infection of 2-week-old AKR mice with the dual-tropic virus DTV-70 induced characteristic changes in the thymus and accelerated leukemia development. DTV-70 inoculation into 14-day-old AKR mice did not change the spontaneous PLC distribution pattern in the tested host organs within 30 days postinfection, nor did it change PLC-specific host requirements for further progression into leukemic cells; however, it enhanced PLC transition to autonomous leukemic cells. The preferential cell tropism of DTV-70 for target cells (prothymocytes) among bone marrow and young spleen cells rather than for thymocytes was also demonstrated in an in vitro-in vivo test. The dual tropic virus may act as a promoter on preexisting PLC (present mostly among bone marrow cells) by enhancing their ability to progress into autonomous leukemic cells.     

Suppression of tumor metastasis by manganese superoxide dismutase is associated with reduced tumorigenicity and elevated fibronectin

We have examined the tumorigenicity and the level of extracellular matrix proteins in fibrosarcoma cells expressing low (SOD-L) and high (SOD-H) MnSOD activities as well as the fibrosarcoma cells transfected with the selectable marker alone (NEO). When the cells derived from each tumor cell line were injected into syngeneic mice., the number of tumor cells required to make a tumor in one-half of the mice (TD50) was markedly increased in MnSOD-transfected cells. The decrease in the tumorigenicity of the MnSOD-transfected cells was associated with an increase in the fibronectin level. These results support the hypothesis that MnSOD is a new type of tumor-suppressor gene.     

肿瘤细胞固有活性氧水平与氧化砷促调亡易感性的关系

目的：研究肿瘤细胞株固有的活性氧(reactive oxygen species,ROS）水平与肿瘤细胞对三氧化二砷（以下简称氧化砷）促调亡的易感性之间的关系。方法：用小剂量（2umol/L）氧化砷作用于人白血病细胞和食管癌细胞各一对（NB4、U937和EC/CUHK1、EC1867）,以证实砷剂促凋亡敏感性在NB4和U937之间的差异以及在EC/CUHK1和EC1867之间的差异。然后在不加砷剂情况下,用活性氧捕获剂双氢罗丹明123（DHR123）孵育细胞（DHR123在细腻内被ROS氧化为发出荧光的罗丹明123,Rh123）,通过流式细胞仪检测细胞内Rh 123的荧光而测得细胞内固有的ROS水平。结果：2umol/L氧化在NB4和EC/CUHK1造成明显凋亡,而在U937和EC1867未造成明显凋亡。对氧化砷敏感的NB4细胞的固有活性氧水平比不敏感的U937细胞高,对氧化砷敏感的EC/CUHK1细胞的固有活性氧水平比不敏感的EC1867细胞高。结论：肿瘤细胞固有ROS水平的差异与肿瘤细胞对三氧化二砷诱导调亡的易感性有关。     

Acquisition of sensitivity to exogenous fibronectin by friend leukemia cells correlates with reduction of their tumorigenic potential

Strongly adhesive, highly flattened clones (FF clones) can be selected from Friend leukemia cells (FLC) cultivated on top of monolayers of human embryonic fibro-blasts (HEF). The flattened phenotype of FF clones is stable during cell replication either in soft agar or in vivo. With the number of passages of FLC on HEF the fibronectin (FN) sensitivity of FF clones increases with a proportional reduction of their tumorigenicity. The FN-sensitivity was defined as the ability of a certain dose of FN to flatten 50% of cells of a given FF clone. Tumorigenicity was defined as the number of FF cells able to give palpable tumors, in 50% of inoculated mice. Exogenous FN does not modify the duplication time of FF clones but strongly influences their growth behavior. In the presence of FN, the growth rate of FF cells is controlled by the size of the growth area available. Highly FN-sensitive FF cells grown on FN-coated substrata die at confluency while FF cells not adherent to substrata escape cell death and grow in suspension. We conclude that the high intrinsic FN-sensitivity of FF cells and FN availability at the site of tumor inoculation could be responsible for the reduced tumorigenicity of highly flattened FF clones.     

Killing of leukemic cells with a BCR/ABL fusion gene by RNA interference (RNAi)

Short 21-mer double-stranded RNA (dsRNA) molecules have recently been employed for the sequence-specific silencing of endogenous human genes. This mechanism, called RNA interference (RNAi), is extremely potent and requires only a few dsRNA molecules per cell to silence homologous gene mRNA expression. We used dsRNA targeting the M-BCR/ABL fusion site to kill leukemic cells with such a rearrangement. Transfection of dsRNA specific for the M-BCR/ABL fusion mRNA into K562 cells depleted the corresponding mRNA and the M-BCR/ABL oncoprotein. This was demonstrated by real-time quantitative PCR and Western blots. The BCR/ABL knockdown was accompanied by strong induction of apoptotic cell death. Leukemic cells without BCR/ABL rearrangement were not killed by M-BCR/ABL-dsRNA. In addition, to corroborate the extraordinary sequence specificity of RNAi, we designed another RNA oligo matching the M-BCR/ABL fusion site but having two point mutations within its central region. We show that these two point mutations abolished both p210 reduction and induction of apoptosis in K562 cells. Finally, we compared leukemic cell killing by RNAi to that caused by the ABL kinase tyrosine inhibitor, STI 571, Imatinib. For full induction of apoptosis, dsRNA targeting M-BCR/ABL required 24 h more than Imatinib. This may be caused by the relatively long half-life of the BCR/ABL oncoprotein, which is not targeted by the RNAi mechanism, but is affected by STI 571. When we applied ds M-BCR/ABL RNA and STI 571 in combination, we did not observe a further increase in the induction of apoptosis. Nevertheless, these data may open a field for further studies towards gene-therapeutic approaches using RNA interference to kill tumor cells with specific genetic abnormalities.     

Genetic reduction of matriptase-1 expression is associated with a reduction in the aggressive phenotype of prostate cancer cells in vitro and in vivo

PURPOSE: Matriptase-1 has been implicated as playing an important role in various types of cancer progression through many different cancer related pathways. In the current study we assessed the efficacy of targeting matriptase-1 using ribozyme technology in vitro and in vivo. EXPERIMENTAL DESIGN: Matriptase-1 expression was reduced in the PC-3 and DU-145 cell line using hammerhead ribozyme transgenes. In vitro assays were set up to assess changes in growth, invasion, adhesion and migration in these cells. In vivo tumour development model was also used to examine the efficacy of targeting matriptase-1 in a living environment. RESULTS: The in vitro results suggest an overall reduction in the aggressive nature of the two cell lines (PC-3 and DU-145) when matriptase-1 levels are reduced, with properties such as growth, invasiveness and migration all being reduced (in most cases a greater than 50% reduction in migration and invasion compared to the control was observed), though strangely an increase in adhesion is seen in the PC-3 knockout. The in vitro data is strongly backed up by the results of the in vivo work which demonstrates matriptase-1 deficient cells have a substantially reduced ability to grow and develop in vivo compared to control cells when explanted into nude mice, with significant differences in growth and development (P < or = 0.05) being seen after 7 days, and highly significant differences (p < or = 0.001) after 15 days. CONCLUSIONS: Together this data strongly implicates matriptase-1 as playing a vital role in the aggressive nature and progression of prostate cancer.     

PMA-induced reduction in invasiveness is associated with hyperphosphorylation of MARCKS and talin in invasive bladder cancer cells

Protein kinase C (PKC) plays a critical role in signal transduction for a variety of cell activation processes. Enhanced PKC activity is often found in cancer cells that show marked invasive and/or metastatic potential. Thus, a specific PKC inhibitor may serve as a tool to reduce invasive or metastatic potential of cancer cells. We show here that phorbol 12-myristate 13-acetate (PMA), a PKC activator, also reduces invasiveness of EJ invasive transitional carcinoma cells. PMA-induced reduction in invasiveness was parallel with inhibition of cell motility. PMA neither induced E-cadherin expression nor augmented cell-matrix adhesion of EJ cells. PMA caused retraction of microspikes from the rim of the cells and consequently rounding of the cellular rim, and the disappearance of microfilaments from the cytoplasm. PMA at 10⁻⁷ M, at which concentration the motility of EJ cells was completely inhibited, down-regulated PKC activity over 5 hr after transient translocation of PKC activity to the membrane fraction. At the same time, PMA induced hyperphosphorylation of MARCKS and talin. During the process of cell movement, actin-binding proteins are in a cycle of phosphorylation and dephosphorylation. Once this cycle is interrupted, cells can no longer maintain the dynamics of cytoskeletal structure. We suggest that retention of the hyperphosphorylated state of MARCKS and talin is responsible for the mechanism(s) by which PMA produces inhibitory activity against invasiveness of EJ cells. Int. J. Cancer 75:774–779, 1998.© 1998 Wiley-Liss, Inc.     

High expression of shMDG1 gene is associated with low metastatic potential of tumor cells

Metastasis is the primary cause of mortality associated with cancer. Molecular mechanisms leading to metastatic spread are poorly studied. To get a better understanding of this process, we compared the gene expression pattern of two isogenic cell lines, HET-SR and HET-SR1 (Rous Sarcoma Virus-transformed embryo hamster fibroblasts) with different metastatic activity using the differential display technique. A novel cDNA of hamster gene shMDG1 (Syrian hamster homologue of microvascular differentiation gene 1), which had 94% homology with rat MDG1 gene, was identified. Expression of shMDG1 was increased in low metastatic HET-SR cell line in comparison to high metastatic HET-SR1. Sequence analysis of the ORF of shMDG1 gene showed that it belongs to the DnaJ/heat-shock proteins of 40 kDa (HSP40) chaperones family, considered to function as a cochaperone of HSP70 family. In order to confirm involvement of shMDG1 in metastasis, we injected parental and shMDG1 overexpressed cells into animals. We showed that overexpression of the shMDG1 gene significantly diminished the metastatic activity of both HET-SR and HET-SR1 cells. The shMDG1-induced repression of metastasis was not connected with alterations in cell proliferation and motility in vitro, but correlated well with a decrease in content of the Asn-linked beta1-6 branched oligosaccharides on cell surface.     

N-glycosylation of glucose transporter-1 (Glut-1) is associated with increased transporter affinity for glucose in human leukemic cells

To elucidate the role of N-glycosylation in the functional activity of the universal glucose transporter, Glut-1, we investigated effects of the N-glycosylation inhibitor, tunicamycin, on glucose transport by human leukemic cell lines K562, U937 and HL60. Treatment with tunicamycin produced a 40-50% inhibition of 2-deoxyglucose uptake and this was associated with a 2-2.5-fold decrease in transporter affinity for glucose (Km) without a change in Vmax. Leukemic K562, U937 and HL60 cells expressed Glut-1 transporter protein. With K562 cells Glut-1 appeared as a broad band of 50-60 kDa, whereas with U937 and HL60 cells a diffuse band was observed at approximately 55 kDa. Treatment of K562 cells with tunicamycin for 18 h, resulted in extensive loss of the 50-60 kDa glycoprotein, appearance of a 30-40 kDa band and increased staining of a 45 kDa band. With U937 cells, tunicamycin treatment resulted in the appearance of a 30-40 kDa band and increased staining of a 45 kDa band. With HL60 cells loss of the 55 kDa Glut-1 band was observed and a band of 45 kDa appeared. Tunicamycin-treatment resulted in 75-90% inhibition in [3H]mannose incorporation but only 20-25% inhibition in [3H]thymidine and [3H]leucine incorporation. In contrast, tunicamycin had little effect on the viability and MTT responses of the cells used. These results suggest that in leukemic cells N-glycosylation of Glut-1 plays an important role in maintaining its structure and functional integration.     

Melanoma metastasis is associated with enhanced expression of the syntenin gene

Primary cutaneous melanomas and melanoma metastases were examined for differential gene expression using subtractive suppression hybridization in a search for any genes associated with metastasis. Generating a subtracted library of candidate genes up-regulated in melanoma metastases, this library contained 8 different cDNAs, among them a cDNA fragment of the syntenin gene which was overexpressed in further independent melanoma resection specimens on cDNA Southern blots when compared to acquired melanocytic nevi from which melanomas are known to arise. Upon immunohistochemistry, the syntenin protein expression was detected in the cytoplasm of primary cutaneous melanomas and melanoma metastases. Melanoma metastases exhibited higher proportions of tumour cells positive for syntenin immunostaining in comparison to acquired melanocytic nevi or non-metastasizing primary melanomas which was statistically significant. In addition to melanoma, gastric cancer tissues exhibited a higher syntenin mRNA expression on a matched tumour/normal cDNA array than their normal counterparts which was statistically significant. Altogether, we here first describe the detection of the syntenin protein in resection specimens of melanocytic lesions. We conclude that melanoma metastasis is associated with increased expression of the syntenin gene which may participate in signal transduction and cell adhesion via the multifunctional protein-binding properties of its tandem PDZ domains.