Stimulation of natural killer cell activity and inhibition of proliferation of various leukemic cells by purified human leukocyte interferon subtypes

One of several human leukocyte interferon subtypes A (LeIF-A), obtained in purified form from a gene cloned in Escherichia coli, stimulated human peripheral blood natural killer cell activity, whereas another human leukocyte interferon subtype D (LeIF-D) had no effect with the use of K562 as target cells. With Daudi as target cells, both LeIF-A and LeIF-D stimulated natural killer cell activity. A hybrid human leukocyte interferon, NH2-terminal 61 amino acids and COOH-terminal 104 residues of LeIF-A and LeIF-D, respectively (LeIF-AD) showed greater stimulation than did LeIF-A, but the stimulation did not exceed that of natural buffy coat interferon. A mixture of equal antiviral units of LeIF-A and LeIF-D was no more effective than was LeIF-A alone. The cloned interferon subtypes showed differential effects on the proliferation of three human leukemic cell lines: Daudi (B-cell lymphoblastoid leukemia); BALL 1 (B-cell acute lymphoblastic leukemia); CCRF-HSB-2 (T-cell acute lymphoblastoid leukemia). Growth of Daudi cells was generally most sensitive to all the interferons tested, LeIF-A, -D, -AD, and a buffy coat preparation; no viable cells remained after 120-hr exposure to 1000-unit/ml doses of the interferons. BALL 1 was relatively resistant to the interferon subtypes tested including LeIF-AD, but this cell lines was very sensitive to a preparation of natural buffy coat interferon. CCRF-HSB-2 showed some sensitivity to all the interferons with greatest sensitivity to LeIF-A (10% of the viable cells were detected after 1000 units/ml exposure for 120 hr). In contrast to the leukemic cell lines tested, human amnion cells (WISH) and the human erythroid leukemia, K562, were resistant to the antiproliferative activity of the interferons.     

Sequential irreversible, actinomycin D-sensitive, and cycloheximide-sensitive steps prior to cortisol inhibition of uridine utilization by P1798 tumor lymphocytes.

Events preceeding the cortisol inhibition of uridine utilization by corticoid-sensitive P1798 lymphocytes have been investigated. When tumor cells were incubated with 1 muM cortisol for 15 min and then washed free of steroid and reincubated in the absence of hormone, the expected decrease of uridine uptake failed to appear 1.5 hr later. In contrast, the removal of cortisol after 30 or 60 min did not prevent subsequent development of the steroid effect. Addition of actinomycin D with cortisol, or 15 min after hormone treatment was started, blocked steroid action. However, when actinomycin D was added 30 or 60 min after the initial exposure to cortisol, hormone-induced depression of uridine uptake was no longer prevented. To study the role of protein synthesis, cycloheximide was added to the tumor cell suspensions at various times after cortisol treatment was started. Cortisol suppression of uridine utilization was blocked when cycloheximide was added with the hormone or 30 min after the start of hormone treatment. Cycloheximide added together with cortisol and washed out with the steroid after 30 min did not prevent subsequent appearance of decreased nucleoside uptake. Hydroxyurea, an inhibitor of DNA synthesis, did not prevent cortisol action, even when present throughout a 2 hr exposure to the steroid. Hormone removal or actinomycin D addition after 1.5 to 2 hr (when uridine uptake was already inhibited about 25%) did not prevent intensification of the steroid effect during a subsequent 1.5- to 2-hr incubation period, while addition of cycloheximide at this time completely prevented its progression. These results suggest aht: (a) cortisol inhibition of uridine uptake by P1798 lymphocytes involves an early irreversible step and appears to require continuing RNA but not protein synthesis during the first 15 to 30 min of hormone action; (b) protein synthesis but not RNA synthesis is required between 30 and 60 min; and (c) continuing protein synthesis but not RNA synthesis or hormone presence is necessary for the preestablished cortisol effect to progress.     

Interferon-γ modulates retinoblastoma gene mrna in monocytoid cells

To study the effect of interferon gamma (IFN-_γ) on the expression of the retinoblastoma (RB) susceptibility gene, we performed Northern-blot analysis on RNA extracted from Wish, HEL and monocytoid cell lines U-937 and THP-I treated with 1,000 IU/ml of recombinant IFN-_γ. In U-937 and THP-I cells, IFN-_γ increased the abundance of RB mRNA. In Wish and HEL cells, co-treatment with cycloheximide was required for IFN-_γ to increase the level of RB mRNA. Pre-treatment THP-I cells with cycloheximide prior to IFN-_y treatment augmented the effects of IFN-_γ on RB gene expression. The effect of IFN-_γ in THP-I cells was observed after 3 hr treatment, being more pronounced after 6 hr and persisting until at least 18 hr, although at a lower level. These results suggest that IFN-_γ regulates the level of RB mRNA by different mechanisms in the different cell types. This cytokine increases the abundance of RB mRNA in monocytoid cell lines, reinforced by prior treatment with cycloheximide. Inhibition of protein synthesis is required in Wish and HEL cell lines before IFN-_γ has an effect on RB gene expression.     

Re‐expression of microRNA‐150 induces EBV‐positive Burkitt lymphoma differentiation by modulating c‐Myb in vitro

Burkitt lymphoma (BL) is a highly aggressive B‐cell lymphoma that includes two forms of BL differing in Epstein–Barr virus (EBV) infection status, EBV‐positive and EBV‐negative. Although many efforts, such as high‐intensity, short‐duration combination chemotherapy, have been devoted to improving therapy for this rapidly proliferating neoplasm, there are still significant treatment‐associated toxicities. Therefore, there remains a need for novel effective therapeutic strategies. MicroRNAs play a role in “fine tuning” the physiological and pathological differentiation process, by which cells can rapidly regulate dynamic events such as cell‐lineage decisions and morphogenesis. This unique miRNA feature shifts the traditional one drug target paradigm to a novel one drug multiple targets paradigm. Here, we found that BL cell lines showed an extremely low expression of microRNA‐150 (miR‐150), and then restored miR‐150 expression at physiologic levels in BL cell lines Daudi, Raji, BJAB, and Ramos. The results showed that re‐expression of miR‐150 reduced proliferation of Daudi and Raji cells. Furthermore, Daudi and Raji, both of which are of EBV‐positive germinal center B‐cell origin, transduced with miR‐150 can be rescued to differentiate toward B‐cell terminal stage. However, no significant changes were observed in BJAB or Ramos cells, which are of EBV‐negative germinal center B‐cell origin. Of note, re‐expression of miR‐150 also resulted in decreasing c‐Myb protein levels. Additionally, c‐Myb knockdown in Daudi and Raji cell lines recapitulated the partial characteristics similar to that caused by re‐expression of miR‐150. Taken together, our findings show that miR‐150 can induce EBV‐positive BL differentiation by targeting c‐Myb.     

Regulation of c-fgr proto-oncogene expression in Burkitt's lymphoma cells: effect of interferon treatment and relationship to EBV status and c-myc mRNA levels

The proto-oncogene c-fgr is expressed in transformed human B lymphoid cell lines and has been reported to be induced in cells infected with the Epstein-Barr virus (EBV). We have compared the levels of c-fgr mRNA in several B cell lines and have examined the effects of interferon-induced changes in growth rate of Daudi cells on the concentration of this mRNA. High levels were found in exponentially growing EBV-positive Raji and Daudi cells but the amounts in B95-8 and P3HR-1 cells (also EBV-positive) were lower than in the EBV-negative cell line Ramos. Growth inhibition of Daudi cells by interferon-alpha is preceded by a reduction in c-fgr expression, with a 56% decrease observed within 6 h. The differences in the amounts of c-fgr mRNA in the various cell lines and in control versus interferon-treated cells are similar to the differences in the c-myc mRNA contents of these cells. These results indicate that c-fgr expression bears little relationship to the EBV status of B lymphoid cell lines but may play a role, in conjunction with c-myc expression, in growth regulation by interferons. Other conditions which influence Daudi cell proliferation, such as treatment with a phorbol ester or growth to high cell density, also inhibit c-fgr expression but to a lesser extent than interferon treatment.     

MDA-7/IL-24对Burkitt淋巴瘤细胞的诱导分化作用

目的:研究黑色素瘤分化相关基因-7(melanoma differentiation associated gene 7,MDA-7)/IL-2对Burkitt淋巴瘤细胞的促分化作用并探讨其作用机制.方法:构建稳定过表达MDA-7/IL-24的人Burkitt淋巴瘤Raji和Daudi细胞株,MTS法检测稳定转染MDA-7/IL-24对Raji和Daudi细胞活力的影响;Transwell小室实验检测转染MDA-7/IL-24对Raji和Daudi细胞侵袭和迁移能力的影响;流式细胞术检测细胞凋亡水平及免疫表型;Western blotting技术检测转染MDA-7/IL-24对Raji和Daudi细胞表达分化相关蛋白Myb、BLIMP1及BCL-6的影响;建立裸鼠Raji细胞移植瘤模型,检测在体内环境中稳定转染MDA-7/IL-24对Raji细胞生物活性的影响.结果:过表达MDA-7/IL-24的Raji和Daudi细胞其增殖(P.＜0.05)、侵袭(P＜0.01)及迁移(P＜0.01)能力均明显下降,但凋亡细胞无明显增加(P＞0.05),表达CD45及CD138的水平均明显增加(P＜0.01),而表达CD10的水平明显下降(P＜0.01).过表达MDA-7/IL-24的Raji和Daudi细胞表达BLIMP1的水平明显增加(P＜0.01),而表达Myb及BCL-6的水平均明显减低(P＜0.01).MDA-7/IL-24过表达组裸鼠模型Raji细胞移植瘤质量明显低于对照组[(1.23±0.21)vs(1.96±0.24)g,P＜0.01].结论:转染MDA-7/IL-24可能通过诱导分化作用抑制Burkitt淋巴瘤细胞的生物活性.     

Evaluation of the binding of radiolabeled rituximab to CD20-positive lymphoma cells: an in vitro feasibility study concerning low-dose-rate radioimmunotherapy with the alpha-emitter 227Th

Radioimmunotherapy (RIT) with the alpha-emitter 227Th is currently under evaluation. 227Th is conjugated to the chimeric anti-CD20 monoclonal antibody rituximab, using the chelator p-isothiocyanato-benzyl-DOTA. In this study, the binding of 227Th-DOTA-p-benzyl-rituximab to three different CD-20-positive lymphoma cell lines, Raji, Rael, and Daudi, were evaluated. Equilibrium and kinetic binding experiments were used to determine binding parameters, including the association and dissociation rate constants, the equilibrium dissociation constants, and the total number of antigens for Raji, Rael, and Daudi cells. There were significant differences between the cell lines with respect to both Kd and the total number of antigens. Rael cells had more than three times as many antigens as the other two cell lines, and the functional Kd found for Rael cells was significantly higher than that found for Raji and Daudi cells. These results were confirmed using flow cytometry. Rituximab was found to be localized in patches on the cell membrane. The findings indicated that 227Th-labeled rituximab has relevant antigen-targeting properties for radioimmunotherapy.     

Effects of interferon-beta in combination with 5-fluorouracil on the growth of esophageal cancer cells in vitro

The possible antiproliferative potency of human recombinant interferon-beta (hIFN-beta) towards ten human esophageal cancer cell lines was examined in comparison with the activity of the factor towards human malignant melanoma cell lines. The cell growth of esophageal cancer cell lines was inhibited by hIFN-beta in a dose- and time- dependent manner. The 50% inhibitory concentrations (IC50) of hIFN-beta on nine cell lines out of ten ranged between 23 to 332 IU/ml of culture medium. The remaining cell line, T.Tn, was less sensitive to the interferon (IC50, 611 IU/ml). Under the same culture conditions, the melanoma cell lines tested differed markedly in their sensitivity to hIFN-beta. When the esophageal cancer cells were treated with 5-fluorouracil (5-FU) in the presence of a low concentration of hIFN-beta, the effectiveness of 5-FU was markedly enhanced. In particular, the rate of growth inhibition of T.Tn cells was more than the added potencies of 5-FU and hIFN-beta indicating that the interferon is an effective biomodulator of 5-FU. All these data suggest that combination therapy with hIFN-beta and the anticancer drug 5-FU would be beneficial for the treatment of carcinoma of the esophagus.     

Effect of human leukocyte interferon on the metastatic lung tumor of osteosarcoma: case reports

Three patients with pulmonary metastases of osteosarcoma underwent treatment with intravenous or intramuscular administration of human leukocyte interferon. In 2 cases, the size of the metastasized tumor mass diminished temporarily six or eight months after interferon treatment, and serum alkaline phosphatase levels decreased to normal. In 1 case, the tumor cell from the pleural exudate 1 could be isolated and cultivated in vitro. When 2,000 IU of interferon was added to the tumor cell culture, marked inhibition of tumor cell growth resulted. In the other case, interferon had no effect on the metastasized lung tumor.     

Effects of human gamma interferon on cell growth, replication of virus and induction of 2'-5'oligoadenylate synthetase in three human lymphoblastoid cell lines and K562 cells

Human alpha interferon (IFN-alpha) and beta interferon (IFN-beta) showed antiviral and anticellular effects on human lymphoblastoid Daudi and P3HR-1 cells, but up to 1,000 units/ ml of gamma interferon (IFN-gamma) showed no such effect. Though a fairly high level of dsRNA-dependent 2'-5'-oligoadenylate synthetase (2-5A synthetase) was found in Daudi cells, treatment of these cells with IFN-alpha and beta enhanced the enzyme level in cells at least four-fold, but IFN-gamma did not show any such effect. Lymphoblastoid Raji cells were insensitive to the antiviral and anticellular activities of IFN-alpha, beta and gamma, but 2-5A synthetase was induced in cells by the treatment with IFN-alpha and beta, though the enzyme level was lower than that found in interferon-treated Daudi cells. Human leukemic K562 cells were completely insensitive to IFN-alpha, beta and at the same time to IFN-gamma with regard to the antiviral, anticellular activities and to the induction of 2-5A synthetase.     

Effects of nIFN β and rifn γ on growth and morphology of two human melanoma cell lines: Comparison between two-and three-dimensional culture

We compared the anti-proliferative effects of natural inter-feron β (nIFN β) and recombinant interferon γ (HFN γ) on 2 human melanoma cell lines, IGRI and SK-Me128, grown in 2-dimensional monolayer and in 3-dimensional spheroid culture. In monolayer culture, growth of both lines was inhibited in a dose-dependent manner by 5-day treatments with IFN in concentrations ranging between I and 5,000 IU/ml. Incubations with 120 IU/ml nIFN βor 25 IU/ml rIFN γ led to a 50% growth inhibition of IGRI cells. A 50% growth inhibition of SK-Me128 cells was obtained with 60 IU/ml nIFN β, whereas even 5,000 IU/ml rIFN γ inhibited the growth of this line by only 30%. Growing these melanoma cell lines in 3-dimensional spheroid culture for 5 days reduced their sensitivity to interferon. Growth inhibition values of 50% were achieved with 3,000 IU/ml rIFN γ or 9,000 IU/ml nIFN β for IGRI spheroids and 10,000 IU/ml nIFN β for SK-Me128 spheroids, while 10,000 IU/ml rIFN γ reduced the growth of SK-Me128 spheroids by only 25%. Outgrowth tests showed that the proliferative capacity after 5-day incubations with IFN was only reduced in IGRI spheroids treated with high doses of nIFN β. The macroscopically observed increased density of interferon-treated spheroids could be confirmed by light microscopy as corresponding to reduced intercellular space in these spheroids. Scanning electron microscopy furthermore showed variations on the surface of IFN-treated spheroids as well as in cellular organization and structures between cells, hinting at a possible involvement of extracellular matrix substances in the reaction to interferons. These findings show that the high anti-proliferative sensitivity of the melanoma cell lines in monolayer culture could not be achieved in spheroid culture. The volume decreases seen in the presence of interferon might be, at least in part, due to an increased packing density in combination with changes in the cellular organization of interferon-treated spheroids, rather than to direct antiproliferative effects.     

Activity of proteolytic enzymes and their inhibitors during proliferation of P-815 mastocytoma cell proliferation in vitro]

Proliferation of mastocytoma P-815 cells in vitro was accompanied by a rise in cathepsin D, elastase- and trypsin-like proteinase activity during 6 hours of culturing and a decline by hour 24. Yet alpha 1-proteinase inhibitor activity was inversely proportional to proteinase concentration. Antiproliferative action of actinomycin D disrupted phase variation of proteinase activity and, consequently, the level of alpha 1-proteinase inhibitor rose after 6 hours of cell culturing while that of alpha 2-macroglobulin--after 48 hr. Antiproliferative effect of actinomycin D was eliminated by reduced inhibitor level brought about under the influence of exogenous trypsin. When trypsin was added cathepsin D activity reached its peak 6 hr later while that of alpha 1-proteinase inhibitor declined. That effect and the actomycin D-proteinase inhibitor mechanism were retained when trypsin and actomycin D were present together. It is suggested that cathepsin D and alpha 1-proteinase inhibitor activity plays a key role in realizing the proliferative potential of mastocytoma P-815 cells.     

Influence of interferon on adriamycin uptake of cultured tumor cells

Effect of human interferon beta (IFN beta) or interferon gamma (IFN gamma) on cellular uptake of adriamycin (ADM) was measured in 3 in vitro-cultured human tumor cell lines. Pre-incubation of tumor cells with 100 IU/ml of IFN beta or gamma for 6 hr resulted in an increased intracellular ADM concentration, but ADM efflux was not affected by the IFNs. The increase in cellular uptake of ADM was in agreement with both DNA and protein synthesis inhibition. 3H thymidine (3H-TdR) incorporation was remarkably inhibited by ADM with pre-treatment of 100 IU/ml of each IFN, and an additive effect of ADM and IFN on cell growth inhibition could be observed. Similar additive inhibition against tumor-cell proliferation in nude mice, which received combined treatment (twice/week) of IFN gamma (5,000 IU/mouse) and ADM (5-20 micrograms/mouse), was also observed. These results indicate that IFN increases cellular influx of ADM, and antiproliferative activity against tumor cells is enhanced by a combination of IFN and ADM.     

L-asparaginase kills lymphoma cells by apoptosis

Microscopic examination of histological sections of lymph nodes from a canine case of malignant lymphoma at 4 h after treatment with L-asparaginase revealed massive destruction of neoplastic cells by what was consistent with apoptosis morphologically. Apoptosis as the mode of cell death after asparaginase treatment was confirmed in a mouse lymphoma cell line (LY-TH) by the characteristic fragmentation of DNA into oligonucleosome-sized pieces and by the morphological changes consistent with apoptosis following treatment in vitro. Applied to these cells, asparaginase was found to be most cytotoxic over the range of 1–10 IU/ml. Even after 4 h of asparaginase treatment at 100 IU/ml, protein synthesis was reduced by only one-half, yet DNA fragmentation reached 40%. Other agents that affect protein synthesis (cycloheximide and actinomycin D) caused apoptosis as well; however, agents (radiation, prednisolone, and VP-16) whose mechanisms are different from inhibition of protein synthesis also caused apoptosis. As such, it seems unlikely that protein depletion per se and/or the elimination of specific shortlived proteins is the triggering event that leads to cell death. It is more likely that the suspension of cellular proliferation commits cells to apoptosis after asparaginase treatment.This work was supported by NIH grants CA-06294 and CA-16672     

S-acetyl-glutathione selectively induces apoptosis in human lymphoma cells through a GSH-independent mechanism.

Reduced apoptosis is associated to cancer development. Agents able to restore the programmed cell death responsiveness of cancer cells are foreseen as potential effective cancer therapies. In this study, we report that a glutathione-S-derivative, S-acetyl-glutathione (Sag), induces significant apoptosis in three human lymphoma cell lines, including Daudi, Raji and Jurkat cells while it had no or little effect on either Hut-78 lymphoma cells or the normal BT lymphocytes. We used Annexin-V FACS analysis and DNA laddering to demonstrate that Sag activated apoptosis in the three sensitive cell lines in a dose- and time-dependent-fashion. Using mercury orange staining and FACS analysis, we showed that Sag generated an intracellular GSH depletion in Daudi, Raji and Jurkat cells but not in Hut-78 or the normal BT cells. These data provide direct evidence that Sag specifically activates programmed cell death in lymphoma cells through, at least in part, a depletion of intracellular GSH rather than an increase, as previously suggested. Because of its selective effect on cancer cells, Sag appears as a promising new lymphoma cell apoptosis inducer with potential clinical value for lymphoma patients.     

Quercetin induces type-II estrogen-binding sites in estrogen-receptor-negative (MDA-MB231) and estrogen-receptor-positive (MCF-7) human breast-cancer cell lines

We show that flavonoids positively regulate type-II estrogen-binding site (type-ll EBS) levels both in MCF-7 (ER-positive) and in MDA-MB231 (ER-negative) breast-cancer cells. Type-ll EBS were measured by a whole-cell assay at 4°C for 2.5 hr using [³H]-estradiol as tracer. In both cell lines the effect of quercetin (Q) was dose-related and already evident after 12 hr of Q treatment. The increase of type-ll EBS levels after Q exposure requires both RNA and protein synthesis, since actinomycin D and cycloheximide completely abolished the stimulatory effect. The ability of flavonoids in inducing type-ll EBS is well correlated with their relative binding affinity for type-ll EBS. The flavonoid-induced enhancement of type-ll EBS levels is accompanied by increased sensitivity of cancer cells to the inhibitory effect of low Q concentrations. Our data suggest that type-ll EBS are ligand-regulated receptors.     

Combination chemotherapy in vitro exploiting glutamine metabolism of human glioma and medulloblastoma

The human glioma-derived cell line D-54 MG and the human medulloblastoma-derived cell line TE-671 have been shown to be sensitive in culture to the pharmacological interference with glutamine metabolism by acivicin, 6-diazo-5-oxo-L-norleucine, and methionine sulfoximine. Using as a guide the multiple contributions of glutamine to the biosynthesis of proteins, purines, and pyrimidines, we now have identified six additional antimetabolites active against these lines in vitro at clinically relevant concentrations. The 50% growth-inhibitory levels of the drugs against D-54 MG in 6-day continuous exposure experiments were: L-asparaginase, 0.057 IU/ml; 5-fluorouracil, 0.5 micrograms/ml; 6-mercaptopurine, 0.8 micrograms/ml; actinomycin D, 0.0007 micrograms/ml; N-phosphonacetyl-L-aspartic acid, 2.3 micrograms/ml; and 5-azacytidine, 0.2 micrograms/ml (3-day exposure. The corresponding 50% growth-inhibitory values in TE-671 were: L-asparaginase, 0.54 IU/ml; 5-fluorouracil, 1.5 micrograms/ml; 6-mercaptopurine, 4.7 micrograms/ml; actinomycin D, 0.00044 micrograms/ml; N-phosphonacetyl-L-aspartic acid, 4.5 micrograms/ml; and 5-azacytidine, 0.49 micrograms/ml. Dipyridamole up to 10 micrograms/ml was inactive against both lines. The isobologram method was used to evaluate the effectiveness of several two-drug combinations which were biochemically designed. The sums of the optimal fractional inhibitory concentrations for the pairs were: acivicin plus L-asparaginase, 0.14; acivicin plus methionine sulfoximine, 0.40; 6-diazo-5-oxo-L-norleucine plus methionine sulfoximine, 0.60; acivicin plus 6-mercaptopurine, 1.0, all in TE-671; and acivicin plus 5-fluorouracil, 0.79, in D-54 MG. Our findings suggest that an antimetabolite regimen exploiting glutamine sensitivity might improve the chemotherapy of some human gliomas and medulloblastomas.     

Interferon-induced nuclear DNA alterations in malignant carcinoid tumors in vivo

Tumor cell specimens were obtained by ultrasonically guided percutaneous needle liver biopsy from 23 patients with metastatic small intestinal carcinoid tumors. The patients were admitted to the hospital for antitumor therapy (streptozocin, 5-fluorouracil, and leukocyte interferon). The tumor cell samples were used for DNA cytofluorometry. All tumors (12 cases) from untreated patients exhibited diploid DNA stem lines with low proliferative activity. The mean number of tetraploid cells was 8%. Altered nuclear DNA records were obtained in tumors of 8 patients, all of which were treated with leukocyte interferon at 3-6 X 10(6) IU/day for 4-32 months. The DNA records in these tumors varied, but they were mainly in the range between diploid and tetraploid values. In some cases several nuclear DNA peaks were observed within the limits for the cell cycle. It is assumed that the interferon treatment was responsible for the development of altered DNA cell lines because: 1) all untreated tumors yielded regular diploid DNA histograms, 2) interferon was the only drug administered to all patients with tumors displaying altered DNA records, and 3) modal diploid DNA values were observed prior to the interferon treatment in some patients. It is suggested that interferon in vivo blocked carcinoid tumor cell populations in different phases of the cell cycle. This result might explain the nuclear DNA profiles registered in the treated patients as well as the therapeutic effect obtained by interferon.     

The down-regulation of alpha-interferon receptors in human lymphoblastoid cells: relation to cellular responsiveness to the antiproliferative action of alpha-interferon

Human lymphoblastoid cell lines (Daudi, Daudi subclones, Raji and MOLT-4) were compared for sensitivity to the antiproliferative action of alpha-interferon (IFN-alpha) and down-regulation of IFN-alpha receptors. IFN-sensitive and IFN-resistant cell lines have similar numbers (2-4000/cell) of high affinity (20-75 pM) IFN-alpha receptors. Treatment of IFN-sensitive cells with low concentrations (3-10 pM) of IFN-alpha results in low receptor occupancy and nearly complete (greater than 95%) down-regulation of cell surface IFN-alpha receptors within 5 h. Treatment of resistant cells with higher IFN concentrations (30 pM) only results in partial (approximately 60%) receptor down-regulation that is directly related to receptor occupancy. Receptor-receptor interactions, induced by IFN-alpha binding, may account for the enhanced down-regulation of IFN-alpha receptors in IFN-sensitive cells. Such interactions apparently do not occur in IFN-resistant lymphoblastoid cell lines.     

The combined effects of high-energy shock waves and cytostatic drugs or cytokines on human bladder cancer cells.

The effects of shock waves generated by an experimental Siemens lithotripter in combination with cytostatic drugs or cytokines on several bladder cancer cell lines were examined in vitro. Proliferation after treatment was determined with the 3-4,5-dimethylthiazol-2,5 diphenyl tetrazolium bromide assay. Dose enhancement ratios were calculated for each drug and each shock wave application mode in order to characterise the sensitising effect of shock wave pretreatment. The influence of the time between shock wave and drug treatment as well as the effects of different sequences of shock wave and drug treatment or concomitant treatment were assessed for selected combinations of cell lines and drugs. It was found that shock wave treatment could render certain cell lines more susceptible to subsequent cis-platinum, mitomycin C or actinomycin D incubation. Cell lines sensitive to tumour necrosis factor alpha or interferon alpha were further sensitised to these cytokines by shock wave pretreatment. The enhanced sensitivity to cis-platinum and actinomycin D decreased rapidly during the first hours after shock wave treatment. The antiproliferative effect was most pronounced after concomitant shock wave and drug treatment. The sensitisation to interferon alpha diminishes more slowly after shock wave exposure. From the results presented in this study it is concluded that transient shock wave-induced permeabilisation of cell membrane not only enhances drug efficiency, but also causes damage to cell organelles and alterations in cellular metabolism.