Current concepts and newer developments in the treatment of malignant gliomas.

Primary malignant brain tumors account for only 2% of all adult cancers but they cause a disproportionately high cancer-related disability and death. Survival of malignant glioma patients has changed only modestly over the past three decades despite the emergence of new treatment strategies for these tumors. In this review, we describe the standard treatment modalities for malignant glioma, which include surgery, radiation therapy and chemotherapy, as well as the status of novel therapies that have been developed to target various aspects of glioma cell biology. We also address this issue of drug delivery as a factor limiting the efficacy of systemic administration of therapeutics and attempts to overcome this barrier. Further progress towards a cure for malignant gliomas will require a greater understanding of the underlying mechanisms driving the growth, and resistance to therapy, of these challenging tumors.

The effects of electromagnetic fields on peripheral blood mononuclear cells in vitro.

OBJECTIVE: A discussion about the adverse effects of electromagnetic waves on the biological life has been ongoing since the discovery of electricity in the 19th century. MATERIALS AND METHODS: The primary objective of this study was to analyze the changes in the cell viability, rates of apoptosis, proliferation indices and the cell surface antigenic structures resulting from 2-, 6- and 24-hour exposure of mononuclear cells isolated from the peripheral blood to 450, 900 and 1784 MHz electromagnetic waves. RESULTS: Data obtained showed that electromagnetic waves didn't have any effect on the cell viability, rates of apoptosis and proliferation index. While electromagnetic waves didn't affect the HLADR and CD11b expression in the peripheral blood mononuclear cells, they decreased the CD11a expression and increased the CD49d expression. CONCLUSION: These data suggest that electromagnetic signals could affect the functional capacity of the peripheral blood mononuclear cells by changing their adhesion ability. Maybe these alterations are the sign of the immune system modulation. More comprehensive studies are needed, involving higher number and more lines of cells (Tab. 6, Fig. 3, Ref. 11).

Active immunotherapy for cancer patients using tumor lysate pulsed dendritic cell vaccine: a safety study.

Cancer vaccine therapy represents a promising therapeutical option. Consistently, with these new treatment strategies, the use of dendritic cell vaccines is becoming increasingly widespread and currently in the forefront for cancer treatment. The purpose of this study was to evaluate the feasibility and safety of tumor lysate-pulsed dendritic cell (DC) vaccine in patients with advanced cancers. For this purpose, eighteen patients with relapsed or refractory cancer were vaccinated with peripheral monocyte-derived DCs generated with GM-CSF and IL-4, and pulsed consequently with 100 microg/ml of tumor lysate before maturation in culture in the presence of IL-1beta, PGE2 and TNF alpha for two days. The first two vaccinations were given intradermally every two weeks while further injections were given monthly. Tumor lysate-pulsed dendritic cell injections were well-tolerated in all patients with no more than grade 1 injection-related toxicity. Local inflammatory response was mainly erythematous which subsided in 48 hrs time. No end organ toxicity or autoimmune toxicity was identified. Clinical responses observed in our study were satisfactory for a phase I clinical study. We observed 4 (22%) objective clinical responses. These responses are significantly correlated with delayed type hypersensitivity testing (DTH) (p < 0.01). The results showed that this active immunotherapy is feasible, safe, and may be capable of eliciting immune responses against cancer.

Chemotherapy influences inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) activity on 3D breast cancer cell line.

Multicellular tumor spheroids (MTS) are three-dimensional structural forms of tumors grown in vitro in the laboratory. In this study, the aim was to determine the regulation of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) expressions on MTS in response to treatment with the commonly used anti-cancer drugs Doxorubicin and Docetaxel. The spheroids were generated using the "liquid overlay" technique. The distribution of both iNOS and eNOS was detected using indirect immunohistochemistry, while the expression of both iNOS and eNOS was measured using Western blots. Additionally, S-phase analysis using 5-bromo-2'-deoxyuridine (BrdU) was done on the MTS after treatment with doxorubicin, docetaxel, and a combination of the two. The Griess method was used to measure nitric oxide (NO) production in the cells. An increase in iNOS immunoreactivity and a decrease in eNOS immunoreactivity were observed after doxorubicin treatment, when compared with the other groups. Furthermore, upregulation of iNOS and downregulation of eNOS were detected in doxorubicin-treated cells using Western blotting. Insignificant iNOS expression was observed in all of the groups, and it was particularly low in the control and drug combination groups. NO production was also found to be significantly high after docetaxel treatment, and cell proliferation decreased after doxorubicin treatment. In conclusion, chemotherapy influences NOS activity differently with the presence of different drugs. The results with iNOS show that doxorubicin is a more effective drug than docetaxel, and a drug combination may play a helpful role in the suppression of tumorigenicity and cancer metastasis. Interestingly, eNOS expression increased after the addition of both docetaxel and the drug combination, and it was found to negatively correlate with the histological grade of the tumor. Therefore, analyzing the expression of both iNOS and eNOS might be very useful for targeting the treatment of breast carcinoma and obtaining better information on prognosis.

Expression of the catalytic and regulatory subunits of protein phosphatase type 2A may be differentially modulated during retinoic acid-induced granulocytic differentiation of HL-60 cells.

To elucidate the regulation of protein phosphatases types 1 (PP1) and 2A (PP2A) during all-trans retinoic acid (ATRA)-induced granulocytic differentiation of HL-60 cells, the phosphatase activity, proteins, and gene expressions of PP1 and PP2A were examined. Treatment with 1 microM ATRA caused an 85% decrease in the PP2A activity in extracts from HL-60 cells, while the PP1 activity was constant. This reduction in PP2A activity appeared to parallel phenotypic and functional changes of HL-60 cells induced by ATRA. Western blot analysis showed that the level of PP2A catalytic subunit (PP2A-C) decreased during the course of ATRA-induced differentiation, whereas expressions of A and B (M(r) 55,000) regulatory subunits of PP2A were relatively unaltered. Expressions of PP1 catalytic subunit isozymes (PP1 alpha, PP1 gamma, and PP1 delta) were not significantly affected by ATRA treatment. Northern blot analysis revealed that mRNA levels of PP2A-C beta and A alpha regulatory subunits were decreased following treatment with ATRA, while levels of PP2A-C alpha and B (M(r) 55,000) alpha regulatory subunit transcripts were relatively constant. Selective down regulation of PP2A-C beta preceded the granulocytic maturation induced by ATRA. Expressions of PP2A-C isoforms and A and B regulatory subunits may be differentially modulated during ATRA-induced granulocytic differentiation of HL-60 cells.

Translocation of protein phosphatase 1 catalytic subunits during 1,25-dihydroxyvitamin D3-induced monocytic differentiation of HL-60 cells.

To elucidate the roles of protein phosphatases type 1 (PP1) and type 2A (PP2A) in 1,25-dihydroxy-cholecalciferol [1,25(OH)2D3]-induced differentiation of HL-60 cells into monocytes, we examined the enzyme activity and the protein and gene expressions of PP1 and PP2A in these cells. Calyculin-A augmented the 1,25(OH)2D3-induced differentiation of the cells. Treatment of the cells with 1,25(OH)2D3 led to a decrease in PP1-like activity in the cytosol fraction, with a concomitant increase in the membrane and nuclear PP1-like activity, as determined when protein phosphatase activity was assayed using myosin light chain as substrate in the presence of 5 nM okadaic acid. Western blot analysis with antibodies specific for PP1 catalytic subunit isozymes (PP1 alpha, PP1 gamma, and PP1 delta) showed that all three PP1 isozymes were expressed but were differentially distributed in each cellular fraction. Subcellular redistribution of PP1-like activity during 1,25(OH)2D3-induced differentiation was mainly attributed to PP1 gamma and PP1 alpha proteins. In contrast, the localizations of PP1 delta and PP2A catalytic and regulatory subunits were not significantly affected by 1,25(OH)2D3 treatment. The gene expressions of PP1 alpha and PP1 gamma appeared to be constant during processes of monocytic differentiation. The correlation between phenotypic and functional changes of HL-60 cells on the one hand and subcellular redistribution of PP1-like activity on the other suggest that the translocations of PP1 alpha and PP1 gamma isozymes may contribute to the 1,25(OH)2D3-induced monocytic differentiation of HL-60 cells.

c-kit gene expression in CD7-positive acute lymphoblastic leukemia: close correlation with expression of myeloid-associated antigen CD13.

Expression of human c-kit proto-oncogene and interleukin-7 receptor (IL-7R) in acute lymphoblastic leukemia (ALL) cells expressing CD7 was examined by Northern-blot analysis and reversed transcription polymerase chain reaction (RT-PCR) assay in relation to the phenotypes. Leukemic cells from four out of 12 CD7+ ALL patients, all of which fulfilled the criteria of ALL in the FAB classification, expressed c-kit genes. Surface CD3 (sCD3) was absent in all of these cases, while cytoplasmic CD3 (cCD3) was found in the two sCD3- cases. CD3 epsilon transcripts were detected in one of the sCD3- cCD3- cases. IL-7R genes were transcribed in the three cases with c-kit gene expression. In addition, there was a good correlation between c-kit gene expression and myeloid associated antigen CD13 positivity of the leukemic cells. None of the patients with c-kit gene expression had mediastinal tumor. Our results show that leukemic cells in a proportion of CD7+ ALL express receptors for cytokines that are secreted by bone marrow stromal cells. Ligands for c-kit genes and IL-7 could play an important role for the regulation of proliferation and differentiation of T-cell progenitors in bone marrow.

Arsenic trioxide-mediated cytotoxicity and apoptosis in prostate and ovarian carcinoma cell lines.

We studied the effect of arsenic trioxide (As2O3) on prostate and ovarian carcinoma cell lines. As2O3 has been shown to be effective in leukemia, and acute promyelocytic leukemia in particular, both in vitro and in vivo. As model cell lines, we used DU145 and PC-3 for prostate cancer and MDAH 2774 for ovarian cancer. New modalities of treatment are essential in these kinds of cancers, which produce a high death toll. The 3-(4,5-dimethyl-thiazoyl-2-yl)-2,5-diphenyl-tetrazolium bromide assay was used to evaluate cytotoxicity. Flow cytometric analysis and mono-oligo nucleosome detection-based ELISA were used to determine the apoptosis. Isobologram analysis was used to evaluate synergism and/or the additive effects of As2O3 and conventional chemotherapeutic agents. We clearly demonstrated that As2O3 has significant cytotoxic effect on both prostate and ovarian carcinoma cell lines. The dose range of As2O3 in all three cell lines was approximately 10(-6) M. The mechanism underlying cytotoxicity of As2O3 was shown to be apoptosis. The experiments by butylated hydroxyanisole showed that the cytotoxic effect of As2O3 was not through superoxide generation. There was no synergism, but the additive effects of As2O3 were demonstrated with cisplatin, adriamycin, and etoposide. We strongly suggest that As2O3 alone or in combination with conventional chemotherapeutic agents be evaluated further as a new agent for the treatment of prostate and ovarian cancers.

Decreased expression of protein phosphatase type 2A in HL-60 variant (HL-60RAr) cells resistant to induction of cell differentiation by all-trans retinoic acid.

To evaluate the molecular basis for susceptibility of the cell differentiation induced by all-trans retinoic acid (ATRA), we examined biochemical activities and expression of protein phosphatases type 1 (PP1) and type 2A (PP2A) from HL-60 cells that are susceptible to differentiation induced by ATRA and HL-60RAr cells, HL-60 variant cells that are resistant to such induction. One nM of calyculin-A (CAL-A) achieved the enhancement of granulocytic differentiation in ATRA-treated HL-60 (1 microM) cells. ATRA exerted no differential action in HL-60RAr cells, but when used in combination with CAL-A, the differential activity was partly resumed at functional and phenotypic levels without change in morphology. The phosphatase activity in the cytosol from HL-60RAr cells was 50% of that from parental HL-60 cells, but the enzyme activities in either membrane or nuclear fractions showed similar values. The decreased phosphatase activity in the cytosol of HL-60RAr cells was mainly due to the decreased expression of the PP2A catalytic subunit. This low level of PP2A protein was reflected at a relative deficiency in expression of the PP2A beta gene in HL-60RAr cells. The exposure to 1 microM ATRA resulted in downregulation of PP2A catalytic subunit protein in HL-60 cells, but ATRA did not affect PP2A expression in HL60RAr cells. Both cell lines expressed the proteins of each PP1 catalytic subunit isozyme (i.e., PP1 alpha, PP1 gamma, and PP1 delta) at comparable levels. ATRA treatment had no effect on the levels of PP1 isozymes. Our results show a correlation between the extent of PP2A expression and the response of HL-60 and HL-60RAr cells to the differentiative effects of ATRA.

Up-regulation of protein serine/threonine phosphatase type 2C during 1 alpha,25-dihydroxyvitamin D3-induced monocytic differentiation of leukemic HL-60 cells.

Treatment with 20 nM 1 alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) caused a progressive increase in the activity of Mg(2+)-dependent protein serine/threonine phosphatase type 2C (PP2C) in subcellular fractions of HL-60 cells, whereas PP2C activity was relatively constant throughout all-trans retinoic acid-induced (1 microM) granulocytic differentiation. The increase in PP2C activity appeared to parallel the 1,25(OH)2D3-induced phenotypic and functional changes in HL-60 cells. Immunoblot and Northern blot analysis indicated that the increase in PP2C activity corresponded to the increased expression of PP2C protein, which was preceded by an increase in the level of mRNA for PP2C beta. No mRNA for PP2C alpha was detected in resting or 1,25(OH)2D3-stimulated HL-60 cells. These results suggest that the increased expression of PP2C is related with the 1,25(OH)2D3-induced monocytic differentiation of HL-60 cells.

Down-regulation by retinoic acid of the catalytic subunit of protein phosphatase type 2A during granulocytic differentiation of HL-60 cells.

Activity of protein phosphatase measured in the absence of divalent cations was decreased by 50% during all-trans retinoic acid (ATRA)-induced HL-60 cell differentiation into the granulocytic phenotype. Treatment of HL-60 cells with ATRA led to a dramatic decrease in the amount of protein phosphatase type 2A (PP2A) protein, whereas that of protein phosphatase type 1 (PP1) protein was relatively constant, as detected by immunoblotting with antibodies specific to PP1 and PP2A. The decreased phosphatase activity may be mainly due to a decrease in the expression of the PP2A protein. The mRNA level of PP2A beta was markedly decreased within 5 h after addition of ATRA, but there was only a slight increase in the mRNA level of PP2A alpha. Selective down-regulation of PP2A beta mRNA clearly preceded the cell differentiation induced by ATRA treatment. Thus, PP2A is down-regulated during ATRA-induced differentiation of HL-60 cells into granulocytes.

Differential association of protein Ser/Thr phosphatase types 1 and 2A with the cytoskeleton upon platelet activation.

The association of protein Ser/Thr phosphatase type 1(PP1) and type 2A (PP2A) with the cytoskeleton (Triton X-100 insoluble residue) during human platelet activation was investigated. In unstimulated platelets, 40% of total PP1-like activity was present in the Triton-insoluble cytoskeleton, while only 10% of the total PP2A-like activity was present in this fraction. Stimulation with 1 U/ml thrombin produced a 1.8-fold increase in PP1-like activity and a 7-fold increase in PP2A-like activity, respectively, in the cytoskeletal fraction, under aggregating conditions. Immunoblot analysis revealed that thrombin treatment increased association of PP1 catalytic subunit isozymes (PP1 alpha, PP1 gamma, PP1 delta) and PP2A catalytic subunit with the cytoskeleton, with concomitant decrease of these enzymes in Triton-soluble fractions. The amounts of cytoskeleton-associated PP1 and PP2A depended on the dose of thrombin which could activate platelets. Agonist-induced redistribution of PP1 and PP2A into the cytoskeleton was inhibited by OP-41483 (a prostaglandin I2 analog). Interaction of PP2A with cytoskeletal proteins strongly correlates with aggregation, whereas the association of PP1 with cytoskeleton can be detected upon platelet activation, even in the absence of aggregation. Co-extraction of protein kinase C and myosin light chain kinase with the cytoskeleton eventually translocated to the cytoskeleton, but only during aggregation. These results suggest that differential translocation of PP1 and PP2A to the cytoskeleton is involved in platelet activation, and their association with cytoskeletal proteins may regulate phosphorylation levels together with protein kinases in platelets.

Augmentation of methylprednisolone-induced differentiation of myeloid leukemia cells by serine/threonine protein phosphatase inhibitors.

To elucidate the roles of serine/threonine protein phosphatases type 1 (PP1) and type 2A (PP2A) in methylprednisolone-induced differentiation of HL60 cells into granulocytes and K562 cells into monocytes, we examined the effect of serine/threonine protein phosphatase inhibitors, okadaic acid and Cal-A on the proliferation/differentiation of HL60 and K562 cells. Okadaic acid and Cal-A augmented methylprednisolone induced granulocytic differentiation and cell death of HL60 cells and monocytic differentiation and cell death of K562 cells in different dose ranges, respectively. These data suggest an important role of PP1 and PP2A in the mechanism leading to differentiation of leukemic cells.

Forskolin potentiates G-CSF-induced proliferation of a murine myeloblastic leukemia cell line.

The role of the cAMP/A-kinase signaling pathway in G-CSF dependent proliferation of murine myeloblastic NFS-60 cells was investigated. G-CSF treatment resulted in a rapid and transient elevation of cAMP content of NFS-60 cells. G-CSF treatment of NFS-60 cells also resulted in the activation of A-kinase parallel to the increase in cAMP concentration. A low concentration (0.2-10 nM) of forskolin augmented the G-CSF-dependent cell proliferation, although forskolin by itself had no effect on NFS-60 cell growth. Forskolin did not affect the IL-3-induced proliferation of this cell line. Addition of forskolin resulted in further increases in the cAMP level, activation of A-kinase in NFS-60 cells stimulated by G-CSF. Proliferation of NFS-60 cells by G-CSF, but not by IL-3, was blocked by the axial diastereoisomer of adenosine 3',5'-phosphorothioate (Rp-cAMPS), a competitive cAMP antagonist. KT-5720(8R*,9S*,11S*)-(-)-9-hydroxy-9-n-hexyloxy-8-methyl-2, 3, 9, 10-tetrahydro-8, 11-epoxy-1H, 8H, 11H-2,7b, 11a-triazadibenzo(a,g) cycloocta(c,d,e)trinden-1-one), an A-kinase inhibitor, inhibited the G-CSF-dependent proliferation. These findings suggest that activation of the cAMP/A-kinase signaling pathway may be involved in G-CSF-mediated cell proliferation of NFS-60 cells, whereas IL-3-dependent proliferation is not mediated in such a manner.

Desmopressin usage in elective cardiac surgery.

BACKGROUND: Desmopressin acetate (DDAVP) has been implicated as a promising agent to reduce blood loss in patients undergoing cardiopulmonary bypass. METHODS: The effects of intraoperative desmopressin were studied in 66 patients undergoing coronary artery bypass grafting, randomized equally into desmopressin and control groups. The desmopressin group received 0.3 microg/kg desmopressin at the end of cardiopulmonary bypass. RESULTS: Fibrinogen level of both groups significantly reduced at postoperative 2nd hr, whereas a significant rise was observed at postoperative 24th hr with an intergroup difference favoring the control group (p=0.0307). In the desmopressin group, the activation time of factor VIII shortened during the whole postoperative period being significant (p=0.0127) at postoperative 24th hr. Postoperative von Willebrand factor (vWF) levels of the desmopressin group were significantly higher than the preoperative ones. The control group did not show such important changes in factor VIII and vWF measurements. Platelet aggregation times of both groups prolonged at postoperative 2nd hr. The control group showed significant elevation in ADP induced aggregation time at 2nd hr and significant reductions of platelet activation percentage in response to ADP, epinephrine, collagen and ristocetin at 2nd hr. Postoperative blood loss as well as blood transfusion need did not differ between the two groups. CONCLUSIONS: Despite the improved platelet functions, desmopressin does not seem to have obvious beneficial effects on postoperative hemostasis in patients without any bleeding disorder and undergoing elective cardiac surgery.

Schistosoma mansoni Infection Following Allogeneic Bone Marrow Transplantation.

A case of Schistosoma mansoni infection in a 28 year old male after allogeneic bone marrow transplantation presenting with portal hypertension and gross hematuria is described. Schistosomiasis was confirmed by the discovery of parasites in the feces, together with the failure the patient to respond to multiple antimicrobial and antifungal treatment. After praziquantel administration, toxic or septic shock syndrome evolved and the patients died of acute renal failure on day 39 post-transplant. In this report, we would like to emphasize the importance of pre-transplant stool and urine cultures, and appropriate serologic tests in patients coming from endemic areas. Patients diagnosed with schistosomiasis must be treated at least 3 to 7 weeks before transplantation.

Long-term evaluation of radioisotope synovectomy with Yttrium 90 for chronic synovitis in Turkish haemophiliacs: Izmir experience.

Since 2001 we have performed 105 radioisotope synovectomy (RS) in 65 children and young adults, age ranging from 3 to 25 years with a average of 15 years in Ege University Hospital, Izmir, Turkey. One fourth of cases were below 10 years of age. All patients had severe haemophilia A and B. Ten patients (17 joints) had high responder inhibitor. We prefer to use Yttrium 90 for all joints (5 mCi for knees; 2 mCi for others). The knees were injected in 56 cases, elbows in 24 cases, ankles in 23 cases and shoulders in two cases. Steroid injections were not preferred as the principle drug of choice. Mean follow-up period after procedure was 2 years (range: 6 months to 3.5 years). All inhibitor patients had satisfactory results. The best results were obtained in elbows than knees and ankles. Excellent rates (no bleeding) were observed in grade-II synovitis 84% for knees, 93% for elbows and 50% for ankles. Because of the excellent and good response (bleeding reduction to 75%), rates were 100% for knees and elbows and 92% for ankles. In six cases, repeated injections were given at 6-month interval and all of them had good results. The grading of synovitis seems to be an important parameter than the age of the patient. Even in patients below 10 years of age, outcomes are not satisfactory in all joints with grade-III vs. grade-II synovitis (12% vs. 73%). No serious complications were observed during and after procedure except two cases. A mild and transient inflammatory reaction was observed in the ankle. There was a minimal radioisotope leakage to superficial skin in the elbow. RS seems to be a safe and effective treatment for chronic synovitis causing recurrent joint bleedings.