Ex vivo expansion of highly purified NK cells for immunotherapy after haploidentical stem cell transplantation in children.

BACKGROUND: Allogeneic natural killer (NK) cells are known to show medium to high cytotoxic activity against HLA-nonidentical leukemia or tumor cells. For a possible benefit of post transplant treatment with NK cells after haploidentical stem cell transplantation (haplo-SCT) we developed a clinical scale procedure for NK cell processing observing Good Manufacturing Practice (GMP). METHODS: Allogeneic donor NK cells were selected from 15 unstimulated leukaphereses using two rounds of immunomagnetic T cell depletion, followed by an NK cell enrichment step. CD56 (+)CD3 (-) NK cells were stimulated and expanded in vitro according to GMP. Quality control of NK cell purity, residual T cells and cytotoxic activity was done by multi-coloured flow cytometric analyses. RESULTS: Purification led to an absolute number of 234-1 237 x 10 (6) CD56 (+)CD3 (-) NK cells from leukapheresis harvests with a median purity of 95 % and a 4 to 6(1/2) log depletion of T cells. After two weeks stimulation with IL-2 a five-fold expansion of NK cells with a T cell contamination below 0.1 % was reached. Median cell viability was 95 % after purification and 99 % after expansion. The IL-2 stimulated NK cells showed a highly increased lytic activity against the MHC-I deficient K562 cells compared to freshly isolated NK cells and a medium cytotoxicity against patients' leukemic cells. CONCLUSIONS: Clinical scale enrichment and activation of allogeneic donor NK cells is feasible. High dose NK cell application may be a new treatment option for pediatric patients with leukemia or solid tumors in case of minimal residual disease or unbalanced chimerism post haplo-SCT as we could show for the first three patients .

Cytomegalovirus infection blocks apoptosis in cancer cells.

Recent pathological findings reveal a higher frequency of human cytomegalovirus (HCMV) in tumor cells from different tumors compared with surrounding tissues. Experimental investigations suggest possible supportive effects of HCMV for tumor development and progression. One HCMV effect on tumor cells is the inhibition of apoptosis, leading to the promotion of tumor cell survival. Decreased sensitivity to treatment-induced tumor cell death is a major reason for failure of anticancer chemotherapy. HCMV infection interferes with both the intrinsic and extrinsic cellular apoptosis pathways. HCMV promotes cell survival signaling influencing the tumor suppressor p53 and its relative p73, and stimulates the antiapoptotic Ras/Raf/MEK/Erk- and PI-3K-signaling pathways. Antiapoptotic effects mediated by HCMV are inhibited by antiviral treatment in cell culture. Therefore, a better understanding of the influence of HCMV infection on tumor cell apoptosis might translate into improved anti-cancer therapy.

Selective activity of BS-RNase against anaplastic thyroid cancer.

BACKGROUND: Anaplastic thyroid carcinoma is an aggressive solid tumor that fails to adequately respond to any known chemotherapeutic regimen. The development of effective chemotherapy agents would provide the best chance for long-term survival of patients. MATERIALS AND METHODS: The cytotoxic effects of bovine seminal ribonuclease (BS-RNase) against thyroid carcinoma cell lines with different degrees of differentiation in comparison to non-malignant cells, including human foreskin fibroblasts (HFF) and retinal pigment epithelial cells (RPE), were tested using the MTT dye reduction assay. Induction of apoptosis was demonstrated by annexin V assay and expression of proteins related to apoptosis was investigated by flow cytometry. The antitumoral in vivo effects of BS-RNase were assessed on established xenografts of anaplastic thyroid carcinoma cell line 8505C in nude mice using subcutaneous injections of BS-RNase (12.5 mg/kg once a day, on 20 consecutive days). RESULTS: All the tumor cell lines exhibited marked sensitivity against BS-RNase in comparison to HFF and RPE cells. The greatest growth inhibition was seen in the 8505C line, while IC50 values for papillary (B-CPAP) and poorly-differentiated thyroid carcinoma cells were about 6-fold higher. The cytotoxic action of BS-RNase was associated with induction of apoptosis. Expressions of Fas and Fas-ligand were not influenced by BS-RNase completely, while the down-regulation of Bcl-2 in treated cells was observed. In vivo treatment induced significant tumor regression after the course of 20 consecutive days. No apparent toxic effects of BS-RNase toward non-malignant cells were observed during the in vivo treatment. After cessation of therapy (day 20) tumor volume continued to decrease and the tumor was no longer detectable after 30 days of treatment induction in all animals. CONCLUSION: BS-RNase may have beneficial effects for treatment of aggressive anaplastic thyroid cancer.

Antineoplastic activity of a novel multimeric gemcitabine-monophosphate prodrug against thyroid cancer cells in vitro.

Gemcitabine (Gem) is a deoxycytidine analog that is effective against pancreatic cancer and other malignancies following conversion to the 5'-O-mono-, di- and tri-phosphate forms. We evaluated the cytotoxicity of GemMP[10], a novel multimeric form of 2'-deoxy-2',2"-difluorocytidine-5'-O-monophosphate (gemcitabine monophosphate) against three thyroid carcinoma cell lines established from anaplastic (8505C), papillary (B-CPAP) and poorly-differentiated papillary (BHT-101) cancer. GemMP[10] decreased tumor cell growth at concentrations ranging from 1 to 50 nM. These concentrations were 5- to 10-fold lower than those required for inhibition of tumor cell growth by monomeric Gem. GemMP[10] cytotoxicity occurred via induction of apoptosis. Flow cytometric analysis of GemMP[10] treated cells revealed growth arrest in S-phase. Fas-antigen expression was increased in thyroid cancer cells treated with GemMP[10], whereas Fas-L and Bcl-2 expression were not significantly affected. These results demonstrated that GemMP[10] is a potent cytotoxic agent that serves to induce apoptosis in association with increased Fas expression in cultured thyroid cancer cell lines.

Bovine seminal ribonuclease exerts selective cytotoxicity toward neuroblastoma cells both sensitive and resistant to chemotherapeutic drugs.

BACKGROUND: Bovine seminal ribonuclease (BS-RNase) exerts selective cytotoxicity toward different types of tumor cells. In the present study, we tested the effects of BS-RNase on cultured neuroblastoma (NB) cells resistant to chemotherapeutic agents. The selectivity of the antitumoral activity of BS-RNase was evaluated using cultures of CD34+ hematopoietic stem cells. MATERIALS AND METHODS: Human NB cell lines including IMR-32, UKF-NB-2 and UKF-NB-3 were selected for resistance against vincristine, doxorubicin or cisplatin by exposure to increasing concentrations of the respective drug. The cytotoxicity of the drugs to NB cells was evaluated using a clonogenic assay in a methylcellulose medium. Peripheral blood progenitor cells were obtained from adult healthy donors by positive selection using specific anti-CD34+ antibodies. The toxicity of BS-RNase to CD34+ cells was assessed in the direct clonogenic assay using methylcellulose medium or in ex vivo expansion culture supplemented with hematopoietic growth factors. RESULTS: In the clonogenic assay it was shown that BS-RNase completely inhibits growth of both parental NB cells and their sublines resistant to chemotherapeutic drugs at concentrations (up to 50 micrograms/ml) which have no significant influence on the growth of colony-forming units, granulocyte macrophage and erythroid burst-forming units. Moreover, BS-RNase had no effect on the ex vivo expansion of total hematopoietic cells or of colony-forming cells from CD34+ progenitors. CONCLUSIONS: BS-RNase is a highly efficient agent against NB cells resistant to chemotherapeutic drugs. The lack of toxicity to hematopoietic progenitor cells suggests that BS-RNase is also likely to have tolerable hematopoietic toxicity.

Bovine seminal ribonuclease inhibits in vivo growth of human neuroblastoma cells.

Bovine seminal ribonuclease (BS-RNase) is a homologue of RNase A with specific antitumor activities. It is selectively toxic for neuroblastoma (NB) cells in vitro with no significant effects on the viability of normal human cells. We evaluated the antitumoral effects of BS-RNase on human NB xenografts from UKF-NB-3 cells in athymic (nude) mice. The efficacy of direct intraneoplastic, subcutaneous and systemic delivery of BS-RNase was explored. Systemic administration of BS-RNase (12.5 mg/kg/day intraperitoneally, for 20 days in the course of four weeks) suppressed tumor growth but was not able to induce any cures. Subcutaneous injections (12.5 mg/kg/day for 20 days in the course of four weeks) and intratumoral BS-RNase treatment using the same schedule resulted in complete tumor regression. During 30 days following cessation of treatment no tumor regrowth was observed and animals were free of tumors. Toxic effects of BS-RNase (e.g., on bone marrow and inner organs) were not apparent. This data indicates that BS-RNase fulfills important criteria for a candidate antitumor agent specific for NB.

The antisense oligonucleotide ISIS 2922 prevents cytomegalovirus-induced upregulation of IL-8 and ICAM-1 in cultured human fibroblasts.

Human cytomegalovirus (HCMV) infection is associated with excessive proinflammatory immune responses such as cytokine/chemokine production or upregulation of adhesion molecules on the host cells. It is assumed that these features of HCMV-related immunopathology can not be treated effectively with currently available anti HCMV drugs. In the present study the efficacy of ganciclovir (GCV), foscarnet (PFA), cidofovir (HPMPC), and ISIS 2922, an antisense oligonucleotide complementary to HCMV immediate-early (IE) mRNA, was investigated on HCMV-induced secretion and functional activity of the C-X-C chemokine IL-8 and the expression of the intercellular adhesion molecule-1 (ICAM-1). As compared with mock-infected cells IL-8 production was increased up to 9-fold and ICAM-1 expression up to 4-fold in HCMV-infected fibroblasts. Treatment of infected cells with GCV (40 microM), PFA (200 microM) or HPMPC (2 microM) suppressed completely virus replication as demonstrated by quantification of late (L) antigen expression and infectious virus production. These drugs, however, failed to inhibit IE antigen expression and did not prevent HCMV-induced upregulation of IL-8 and ICAM-1. In contrast, ISIS 2922 (1 microM) suppressed both IE and L antigen expression by 99% and inhibited infectious virus production by 10(4)-fold. Moreover, ISIS 2922 significantly suppressed HCMV-induced upregulation of both IL-8 and ICAM-1 expression on the transcriptional and on the protein level. Our results indicate that ISIS 2922 but not inhibitors of HCMV DNA prevents HCMV-induced upregulation of IL-8 and ICAM-1, both hallmarks of inflammatory processes. Thus, inhibition of HCMV IE expression with ISIS 2922 may be an important strategy for the treatment of HCMV-related immunopathogenesis.

Bovine seminal ribonuclease selectively kills human multidrug-resistant neuroblastoma cells via induction of apoptosis.

Bovine seminal ribonuclease (BS-RNase) is a homologue of RNase A with specific antitumor activity. The cytotoxic action of this agent was examined in human neuroblastoma (NB) cell lines (SK-N-SH and UKF-NB-4) possessing the multidrug resistance (MDR) phenotype and NB cell lines (IMR-32, UKF-NB-1, UKF-NB-2 and UKF-NB-3) without MDR. Although MDR cells expressed large amounts of mdr-1 mRNA, contained functional P-glycoprotein and had 20- to 105-fold lower sensitivities to doxorubicin and vincristine than cells with non-MDR phenotypes, BS-RNase was equally toxic to all NB cells at concentrations employed (0.2 to 100 microg/ml). BS-RNase showed high selectivity for NB cells and was non-toxic to normal fibroblasts and epithelial cells. Ultrastructural investigation and annexin V assay showed that BS-RNase is a powerful inductor of apoptosis. The antitumoral effects of BS-RNase were also demonstrated in vivo using established subcutaneous xenografts in athymic (nude) mice of the MDR-1-bearing UKF-NB-4 cell line. Intratumoral injections (12.5 mg/kg) of BS-RNase over four weeks resulted in complete tumor regression and absence of tumor regrowth over a two-week observation period after cessation of treatment. The results show that BS-RNase selectively kills NB cells by inducing apoptosis and that this agent is active against mdr-1 expressing cells both in vitro and in vivo. BS-RNase fulfills important criteria for a candidate antitumor agent in NB patients with advanced disease.

Human cytomegalovirus infection decreases expression of thrombospondin-1 independent of the tumor suppressor protein p53.

Thrombospondin-1 (TSP-1) is a potent inhibitor of angiogenesis. It has been shown that promoter sequences of the TSP-1 gene can be transactivated by the wild-type tumor suppressor protein p53. As human cytomegalovirus (HCMV) infection inactivates wild-type p53 of various cell types, we investigated whether HCMV infection is associated with reduced TSP-1 production. We found, in conjunction with accumulated p53, that TSP-1 mRNA and protein expression was significantly reduced in HCMV-infected cultured human fibroblasts. To determine whether the observed TSP-1 suppression depends on p53 inactivation, the p53-defective astrocytoma cell line U373MG was infected with HCMV. In these cells TSP-1 expression was also significantly reduced by HCMV infection whereas expression of the p53 mutant variant remained unaltered. In both cell lines the decreased expression of TSP-1 mRNA occurred early after infection (4 hours), indicating that HCMV inhibits TSP-1 transcription during the immediate-early phase of infection before HCMV DNA replication. Inhibition of HCMV DNA synthesis by ganciclovir did not influence TSP-1 reduction whereas the antisense oligonucleotide ISIS 2922, complementary to HCMV immediate-early mRNA, completely prevented the HCMV-mediated TSP-1 suppression. These findings strongly suggest a novel role for HCMV in the modulation of angiogenesis due to p53-independent down-regulation of TSP-1 expression.

Aphidicolin glycinate inhibits human neuroblastoma cell growth in vivo.

Aphidicolin is a fungal derived tetracyclic diterpene antibiotic. It is selectively toxic for neuroblastoma (NB) cells in vitro but has no significant effects on the viability of normal human cells and a variety of other tumor entities. We evaluated the antitumoral effects of the water soluble ester aphidicolin glycinate (AphiG) on established human NB xenografts from UKF-NB-3 cells in athymic (nude) mice. Furthermore, we explored the efficacy of direct intraneoplastic and systemic delivery of AphiG. Systemic administration of AphiG (60 mg/kg intraperitoneally, twice per day on 10 consecutive days) significantly suppressed tumor growth but was not able to induce any cures. In contrast, intratumoral AphiG injections (60 or 40 mg/kg/twice a day for 4 days) induced complete tumor regression. Two weeks after the end of treatment no tumor cells were microscopically detectable. Animals were free of tumor for more than 90 days. Histologic examination of inner organs and bone marrow did not reveal any apparent toxic effects of AphiG. These data strongly indicate that AphiG deserves further evaluation as a specific treatment for neuroblastoma.

Selective cytotoxic activity of a novel ribonucleoside diphosphate reductase inhibitor MDL-101,731 against thyroid cancer in vitro.

The dramatically increased number of new cases of disseminated children thyroid cancer in Belarus after the Chernobyl accident requires development of novel strategies to treat this disease. In addition to conventional therapy using radioiodine, chemotherapy with new effective drugs can be an alternative kind of treatment. We tested the antitumor activity of (E)-2'-fluoromethylene-2'-deoxycytidine (MDL-101,731), a ribonucleoside diphosphate reductase inhibitor against three thyroid carcinoma cell lines established from anaplastic (8505C), papillary (B-CPAP) and poorly differentiated papillary (BHT-101) cancer. MDL-101,371 decreased both cell growth and DNA synthesis of tumor cells at concentrations lower than 100 nM, while the concentrations higher than 5000 nM showed only moderate effects on growth of normal human fibroblasts. The effects of MDL-101, 371 on tumor cells were associated with induction of apoptosis, as demonstrated by DNA fragmentation assay. Flow cytometric analysis of the expression of apoptosis-related genes revealed the increased levels of Fas-antigen, whereas the levels of Bcl-2 were not significantly influenced in thyroid cancer cells treated with MDL-101,731. These results demonstrated that MDL-101,731 is a potent antitumor agent against cultured thyroid cancer cells due to its ability to induce apoptosis in association with increased Fas expression.

Cytotoxicity of L-cycloserine against human neuroblastoma and medulloblastoma cells is associated with the suppression of ganglioside expression.

BACKGROUND: Human neuroblastoma and medulloblastoma express abnormal ganglioside patterns especially GD2 and GM2 which are important for tumour growth. We tested the effects of L-cycloserine (L-CS), a potent inhibitor of synthesis of glycosphingolipids, on the growth, viability and expression of GD2 and GM2 in neuroblastoma and medulloblastoma cells. MATERIALS AND METHODS: The cytotoxic effects of L-CS were tested using the MTT dye reduction assay on four neuroblastoma (IMR-32, SK-N-SH, UKF-NB-2 and UKF-NB-3), two medulloblastoma (D283 and D341) and normal human fibroblasts and epithelial cell lines. In some experiments cytotoxicity of L-CS was tested in the presence of exogenous GD2 and GM2. The expression of GD2 and GM2 was analysed by flow cytometry. The antitumoral effects of L-CS in vivo were assessed on established xenografts of UKF-NB-3 or D283 cells in athymic (nude) mice using systemic administration of the drug (150 mg/kg intraperitoneally, once per day on 20 consecutive days). RESULTS: In vitro experiments revealed that L-CS was toxic for tumour cells at concentrations ranging from 0.5 to 20 micrograms/ml without any significant effects on normal fibroblasts and epithelial cells. L-CS treatment of UKF-NB-3 and D283 cells significantly inhibited expression of GD2 and GM2. The addition of exogenous GD2 and GM2 to culture medium partially prevented cytotoxic effects of L-CS on tumour cells. In vivo treatment resulted in complete tumour regression of UKF-NB-3 xenografts whereas growth of D283 xenografts was reduced by 60%. CONCLUSIONS: L-CS is a selective antitumoral agent for neuroblastoma and medulloblastoma cells with the ability to reduce expression of tumour associated gangliosides. In vivo experiments suggest that L-CS may be effective drug for treatment of neuroblastoma and medulloblastoma.

Proinflammatory potential of cytomegalovirus infection. specific inhibition of cytomegalovirus immediate-early expression in combination with antioxidants as a novel treatment strategy?

We observed the effects of antiviral therapy on CMV and/or oxidative-stress-induced stimulation of proinflammatory molecules including interleukin-8 (IL-8), melanoma growth stimulatory activity-alpha (GRO-alpha) and intercellular adhesion molecule 1 (ICAM-1) using human foreskin fibroblasts. Ganciclovir, foscarnet or cidofovir completely suppressed virus replication, as demonstrated by CMV late (L) antigen production. These drugs did not influence CMV immediate-early (IE) antigen expression and had no effects on CMV-induced cellular changes in IL-8, GRO-alpha and ICAM-1 levels. Phosphorothioate oligonucleotide (ISIS 2922) suppressed both CMV IE and L antigen by 99%. ISIS 2922 completely suppressed CMV-induced upregulation of both chemokines and ICAM-1. Induction of oxidative stress by H(2)O(2) upregulated IL-8 expression. Oxidative stress and CMV infection showed synergistic effects on IL-8 expression. ISIS 2922 only partially inhibited the upregulation of IL-8 in infected cells treated with H(2)O(2), whereas cotreatment with ISIS 2922 and antioxidants inhibited the upregulation almost completely. The results showed that inhibition of CMV IE expression alone or in combination with antioxidants is promising for the treatment of CMV disease.

Azidothymidine resistance of H9 human T-cell lymphoma cells is associated with decreased sensitivity to antitumor agents and inhibition of apoptosis.

Biology of HIV-1 associated neoplasias is modulated by viral and host factors. In addition the development of tumors and their response to therapy may be further influenced by long-term treatment of HIV-1 patients with nucleoside analogs such as AZT (3'-azido-3'deoxythymidine), ddI (2',3'-dideoxyinosine), ddC (2',3'-dideoxycytidine), d4T (2',3'-didehydro-2'3'-dideoxythymidine), and 3TC [(-)-beta-L-2',3'-dideoxy-3'-thiacytidine] alone or in combination. As these compounds can trigger mechanisms involved in chemoresistance, we tested whether prolonged in vitro treatment of H9 cells (T-cell lymphoma) with AZT alters sensitivity of lymphoma cells to antitumor agents used for AIDS-associated malignancies. H9 cells grown for more than two years in medium containing 250 microM AZT developed resistance to the toxic effects of AZT while retaining sensitivity for other nucleoside analogs including ddC or cytosine arabinoside (ARA-C). These cells designated H9rAZT250 were 2 to 10-fold less sensitive to the toxic effects of antitumor agents, including cisplatin (CDDP), vincristine (VCR), doxorubicin (DOX) and etoposide (VP-16), when compared with parental H9 cells. The resistance of H9rAZT250 cells to antitumor agents was associated with inhibition of apoptosis as demonstrated by ultrastructural investigations and DNA-fragmentation assay (ELISA). The expression of the antiapoptotic gene bcl-2 was increased in H9rAZT250 cells while expression of other genes involved in the regulation of apoptosis such as c-myc, p53 and Fas was not changed. These results demonstrate that prolonged in vitro treatment of H9 lymphoma cells with AZT results in the development of resistance to antitumor agents in association with inhibition of apoptosis and increased expression of bcl-2. Therefore AZT long-term treatment of some HIV-1 patients with malignancies may have affected behavior of tumor cells including response to therapy.

Persistent human cytomegalovirus infection induces drug resistance and alteration of programmed cell death in human neuroblastoma cells.

Infection with human cytomegalovirus (HCMV) is a common and generally asymptomatic affection in childhood. Its role in neuroblastoma (NB) patients has not yet been elucidated. As evidence grows that HCMV interacts with apoptotic signaling due to the interaction of HCMV gene products with cellular proteins of apoptotic pathways, we used human NB cell line UKF-NB-2 persistently infected with HCMV strain AD169 to study the effects of long-term HCMV infection on programmed cell death of neuroectodermal tumor cells. The cells designated UKF-NB-2AD169 continued to produce infectious virus in successive subcultures over a period of more than 1 year. Up to 20% of cells expressed viral genes or produced infectious virus after initiation of infection. UKF-NB-2AD169 cells were significantly less sensitive to the cytotoxic agents cisplatinum and etoposide than parental (noninfected) UKF-NB-2 cells. These effects were associated with decreased ability of UKF-NB-2AD169 cells to undergo apoptosis and continuous viral replication. UKF-NB-2AD169 cells showed increased levels of antiapoptosis Bcl-2 protein (up to 12-fold), whereas expression of p53 and c-myc was not changed. Treatment of UKF-NB-2AD169 cells with ganciclovir, abolishing virus production, reestablished sensitivity to chemotherapy, lowered Bcl-2 expression, and facilitated inducibility of apoptosis to the level of the parental cell line. The results demonstrate that persistent HCMV infection confers resistance to cytotoxic agents on neuroectodermal tumor cells and protects from apoptosis, probably due to increased levels of Bcl-2 protein. Hence, it is conceivable that HCMV infection before or during tumorigenesis may contribute in some NB patients to failure of therapy.

Sodium valproate inhibits in vivo growth of human neuroblastoma cells.

Sodium valproate (VPA) belongs to the group of simple branched-chain fatty acids and due its anticonvulsive activity is broadly applied in the treatment of epilepsy. We previously showed that VPA is able to induce cellular differentiation, to enhance immunogenicity and to inhibit proliferation of human neuroblastoma (NB) cells in vitro. Furthermore, we demonstrated that VPA inhibits proliferation, enhances neural cell adhesion molecule expression and decreases CD44 expression of human and rat glioma cells in vitro. In the present study we investigated the antitumoral effects of VPA on established human NB xenografts from UKF-NB-3 human NB cells in athymic (nude) mice. When the animals developed s.c. tumors of about 100 mm3 volume they were treated with 400 or 200 mg/kg/day VPA i.p. At the end of the treatment period (40 days) tumor volumes in animals treated with 400 and 200 mg/kg VPA were about 4- (p < 0.0001) and 2-fold (p < 0.0005) smaller than in the saline-treated control group, respectively. Histological examination of the remnant tumors of treated animals revealed induction of differentiation by induction of stroma-rich tumors and nodules that contained elongated NB cells. Pyknotic nuclei and apoptotic bodies indicated induction of apoptosis. We conclude that VPA is able to abrogate NB growth in vivo and may therefore be useful in the treatment of NB patients.