CVT-E002 stimulates the immune system and extends the life span of mice bearing a tumor of viral origin.

The present study evaluated the dose-related effects of CVT-E002, a proprietary extract of Panax quinquefolius (CV Technologies Inc., Edmonton, AB), in the treatment of a tumor of viral origin, that is, erythroleukemia, in mice. Three treatments including ingestion of 2, 40, and 120 mg/d were compared. The study revealed that the dose of 40 mg/d was particularly effective in stimulating cells mediating nonspecific immunity and extending the life span of tumor-bearing mice. This study represents the first in vivo demonstration of the anticancer efficacy of CVT-E002 in an animal model. CVT-E002 treatment significantly elevated the absolute numbers of natural killer cells and monocytes and reduced the number of tumor cells in the bone marrow and spleen. This study has shown that (1) approximately 30 to 50% of tumor-bearing mice administered CVT-E002 at a dose of 40 mg/d achieved a significantly extended life span, and (2) dosage is critical in producing these ameliorative effects.

Inhibition of viral carcinogenesis by Phyllanthus amarus.

Friend murine leukemia virus (FMuLv) is an acutely oncogenic retrovirus, and its infection leads to erythroblastosis and leukemia in mice. This infection model is used in the search for new antiviral agents. In the present study, the authors have evaluated the potential of an extract of Phyllanthus amarus against FMuLv-induced erythroleukemia in BALB/c mice. Injection of newborn mice with FMuLv resulted in leukemia and animals died due to splenomegaly. Oral administration of P.amarus was found to enhance the life span of leukemia-harboring animals and decrease the incidence of anemia. The authors also performed a series of hematological, biochemical, histopathological, and gene expression analyses to evaluate the effect of P.amarus administration on erythroleukemia initiation and progression. The data obtained indicate that P.amarus administration could significantly decrease the progression of erythroleukemia. Treatment with P.amarus induced the expression of p53 and p45NFE2 and decreased the expression of Bcl-2 in the spleen of infected mice. Histopathological evaluations of the spleen demonstrated that administration of P.amarus decreased the infiltration of leukemic cells into the sinusoidal space when compared with the vehicle treated group. P.amarus is known to inhibit chemically induced neoplasm in different rodent models.The current results indicate that P.amarus has the ability to suppress virally induced cancers as well.

Molecular assessment of the potential combination therapy of cytokines with biphalin and AZT for Friend leukemia virus infection in vitro.

Biphalin, a dimeric enkephalin analog, is under investigation as a potential, long-lasting medication of pain associated with chronic diseases, like cancer or AIDS. The role of cytokines, and splenocytes in anti-Friend leukemia virus (FLV) activity of biphalin, a synthetic opioid, and AZT was investigated in vitro. Mouse splenocytes inhibited FLV replication in Mus dunni (Dunni) cells when they were added to the cell culture. This inhibitory effect of splenocytes also was evident when cells were combined with biphalin and AZT as measured using a focus-forming assay. Under cell-free conditions, recombinant interferon gamma (IFNgamma), interleukin 2 (IL-2) and IL-4 directly inhibited the FLV reverse transcriptase (RT) activity by 27% to 36%. IFNgamma at 0.005 pg to 500 ng inhibited FLVRT activity by 61% to 80%. Acombination of 250 ng IFNgamma and 50 mug biphalin resulted in a 94% reduction of FLVRT activity, as compared with 61% inhibition by IFNgamma alone. The combination of AZT and IFNgamma, IL-2 or IL-4 also induced a stronger suppression of FLV RT activity than either cytokine or AZT used alone. In addition, cloned RT from Moloney murine leukemia virus (MMLV) was directly sensitive to inhibition by biphalin. Thus, the anti-FLV effects of splenocytes in combination with biphalin and AZT in cell culture are likely mediated to a large degree by the direct effect of cytokines. This antiviral activity of splenocytes or cytokines combined with chemotherapy, biphalin, and/or AZT, could be used as a complementary therapy to current approaches for retroviral infection and benefit acquired immunodeficiency syndrome (AIDS) patients. In conclusion, biphalin applied primarily as a new medicine for chronic pain treatment in AIDS patients may play a significant beneficial role as a component of antiviral HIV multidrug therapies.

Engagement of PI-3-kinase mediated protein kinase C zeta activation in protecting Friend cells from ionizing radiation-induced apoptosis.

Murine erythroleukemia cells (Friend) respond to ionizing radiation with the activation and nuclear translocation of p85alpha subunit of phosphatidylinositol-3-kinase (PI-3-kinase) which mediates the downstream activation and nuclear translocation of atypical Protein kinase C zeta (PKC zeta). This event occurs mainly upon high dose of ionizing radiation (15 Gy) and is concomitant to an increase in BrdU incorporation, which probably accounts for a predominant repair DNA synthesis. Following treatment with wortmannin, a relatively specific inhibitor of PI-3-kinase, both an increased number of apoptotic cells and the inhibition of protein kinase C zeta translocation were detected. Altogether the evidence suggests a potential role of the PI-3-kinase/PKC zeta pathway in protecting Friend cells from ionizing radiation-induced apoptosis offering PKC zeta for consideration as possible target of pharmacological treatments.

Novel bicistronic retroviral vector expressing gamma-glutamylcysteine synthetase and the multidrug resistance protein 1 (MRP1) protects cells from MRP1-effluxed drugs and alkylating agents.

We have constructed two retroviral vectors, one expressing multidrug resistance protein 1 (MRP1) alone (SF91MRP) and the other expressing MRP1 and gamma-glutamylcysteine synthetase (gamma-GCS), the rate-limiting enzyme of glutathione biosynthesis (SF91GCS-MRP). We have utilized the hybrid FMEV (Friend mink cell focus-forming/murine embryonic stem cell virus) backbone, previously shown to be efficient in early hematopoietic cells, even when coexpressing two distinct genes. In SF91GCS-MRP, the cDNAs were combined via an internal ribosomal entry site (IRES) sequence from poliovirus, resulting in a bicistronic mRNA produced via the long terminal repeat (LTR). Producer Fly-eco clones were established by trans-infection with vesicular stomatitis virus glycoprotein (VSV-G)-pseudotyped retroviral supernatants. Drug-resistant producer clones were subsequently selected with antimony potassium tartrate, a nonmutagenic MRP1 substrate. By RNA slot-blot and transduction of 3T3 fibroblasts, titers of both SF91MRP and SF91GCS-MRP were found to be greater than 10(6) viral particles/ml. The correct viral integration in the genome was established by Southern blotting. By flow cytometry, both MRP1 and bicistronic clones showed an increase in expression of the MRP1 protein. The bicistronic producer clones, as well as 3T3 cells transduced with SF91GCS-MRP, presented an increase in intracellular glutathione levels, compared with the parental counterparts. Producer cells, 3T3 fibroblasts transduced with either SF91MRP or SF91GCS-MRP, and primary murine myeloid progenitor cells transduced with SF91GCS-MRP were resistant to MRP1-effluxed drugs. However, only bicistronic producers, 3T3 fibroblasts transduced with SF91GCS-MRP, and primary murine myeloid progenitor cells transduced with SF91GCS-MRP were also resistant to alkylating agents. We conclude that the retrovirus SF91GCS-MRP has features that make it a suitable vector to induce bone marrow resistance to multiple classes of chemotherapeutic agents. The strategy of coexpressing gamma-GCS and MRP1 may help to design an effective in vivo selection for various clinical protocols of gene therapy.

Constitutive muscarinic receptors are involved in the growth and differentiation of friend erythroleukemia cells.

Binding experiments with the specific muscarinic ligand [3H]N-methylscopolamine (3H-NMS) have shown the presence of constitutive muscarinic acetylcholine receptors (mAChR) on Friend murine erythroleukemia cells (MELC). Competition experiments with a panel of specific antagonists indicated that the mAChR were predominantly of the M3 subtype. This was confirmed by the rt-PCR analysis of mRNA levels for M1-M5 AChR. Uninduced MELC expressed approximately 2,100 and 1,200 binding sites per cell of growing and resting populations, respectively. The dissociation constant (K(D)) for 3H-NMS was in the picomolar range. The modulation of mAChR upon induction suggested that MELC growth and maturation might be under control of a cholinergic system since mAChR were markedly decreased or virtually absent in MELC induced to terminal division by dimethyl sulfoxide (DMSO) or hexamethylene bisacetamide (HMBA), respectively. In turn, the number of mAChR on MELC committed to polyploidization by colcemid was either increased over or maintained at the control levels when receptor densities were expressed per cell or surface unit (square micrometers), respectively. Moreover, the muscarinic agonist carbachol was found to inhibit MELC differentiation by decreasing by approximately 35% the amount of benzidine-positive (B+) cells in HMBA-induced cultures and, to a lesser degree, also AChE levels. The carbachol effect on erythroid differentiation was reverted by atropine that was found to restore the original amount of B+ cells, while it reduced acetylcholinesterase (AChE) to levels of approximately 66% of control. Such a selective atropine-mediated inhibition of AChE expression was observed also in HMBA-induced MELC supplemented with the antagonist. These results have suggested that mAChR on MELC are functional and might play a role in modulating the expression of either the erythroid or megakaryocytic traits of these cells.

cDNA cloning and functional characterization of the mouse Ca2+-gated K+ channel, mIK1. Roles in regulatory volume decrease and erythroid differentiation.

We have cloned from murine erythroleukemia (MEL) cells, thymus, and stomach the cDNA encoding the Ca2+-gated K+ (KCa) channel, mIK1, the mouse homolog of hIK1 (Ishii, T. M., Silvia, C., Hirschberg, B., Bond, C. T., Adelman, J. P., and Maylie, J. (1997) Proc. Natl. Acad. Sci.(U. S. A. 94, 11651-11656). mIK1 mRNA was detected at varied levels in many tissue types. mIK1 KCa channel activity expressed in Xenopus oocytes closely resembled the Kca of red cells (Gardos channel) and MEL cells in its single channel conductance, lack of voltage-sensitivity of activation, inward rectification, and Ca2+ concentration dependence. mIK1 also resembled the erythroid channel in its pharmacological properties, mediating whole cell and unitary currents sensitive to low nM concentrations of both clotrimazole (CLT) and its des-imidazolyl metabolite, 2-chlorophenyl-bisphenyl-methanol, and to low nM concentrations of iodocharybdotoxin. Whereas control oocytes subjected to hypotonic swelling remained swollen, mIK1 expression conferred on oocytes a novel, Ca2+-dependent, CLT-sensitive regulatory volume decrease response. Hypotonic swelling of voltage-clamped mIK1-expressing oocytes increased outward currents that were Ca2+-dependent, CLT-sensitive, and reversed near the K+ equilibrium potential. mIK1 mRNA levels in ES cells increased steadily during erythroid differentiation in culture, in contrast to other KCa mRNAs examined. Low nanomolar concentrations of CLT inhibited proliferation and erythroid differentiation of peripheral blood stem cells in liquid culture.

STAT5 activation correlates with erythropoietin receptor-mediated erythroid differentiation of an erythroleukemia cell line.

Interaction between erythropoietin (EPO) and its membrane receptor induces the proliferation and differentiation of erythroid progenitors. EPO has been shown to activate the JAK2-STAT5 pathway in various hematopoietic cell lines, although the physiological role of this pathway is unclear. We have previously shown that epidermal growth factor activates a chimeric receptor bearing the extracellular domain of the epidermal growth factor receptor linked to the cytoplasmic domain of the EPO receptor, resulting in proliferation of interleukin-3-dependent hematopoietic cells and erythroid differentiation (globin synthesis) of EPO-responsive erythroleukemia cells. In the present study, we introduced various deletion and tyrosine to phenylalanine substitution in the cytoplasmic domain of the chimeric receptor and expressed these mutant chimeras in an EPO-responsive erythroleukemia cell line, ELM-I-1. Mutant chimeric receptors retaining either Tyr343 or Tyr401 could activate STAT5, judged by tyrosine-phosphorylation of STAT5 and induction of CIS, a target gene of STAT5. These mutants were able to induce erythroid differentiation. However, a chimeric receptor containing both Y343F and Y401F mutations could not activate STAT5 nor induce erythroid differentiation. Thus, Tyr343 or Tyr401 of the EPO receptor are independently necessary for erythroid differentiation as well as STAT5 activation. Moreover, exogenous expression of dominant-negative STAT5 suppressed EPO-dependent erythroid differentiation. These findings suggest that STAT5 plays an important role in erythroid differentiation through the EPO receptor cytoplasmic domain.

Naturin: a potent bio-immunomodifier in experimental studies and clinical trials.

A number of traditional Chinese medicinal herbs have become extremely interesting in the search for potential BRMs in the international medical community, especially in the United States and Japan. Naturin, a new Chinese medical herb produced by XingYa Pharmaceutical Co., Ltd., has enhanced immune response, inhibited tumor metastases and retroviral infection in animal models as well as in clinical studies. The results demonstrated that the inhibition of Natural Killer (NK) and Lymphokine-activated Killer (LAK) cell activity and lymphocyte proliferation was compromised by tumor metastases and retrovirus infection (Murine AIDS), even immunosuppression induced by surgical amputation can be restored by Naturin. It is also shown that Naturin can protect the mice from lethal total body irradiation. These studies indicated that Naturin possesses immunomodulatory effects in vivo for a broad range of stresses. The results of the clinical studies on Naturin have demonstrated: (a) significantly improved symptoms of patients, including MDS, acute and chronic leukemia, aplastic anemia, lung cancer, and association with the increased number and percentage of CD4 (Helper T-cell) which have been reduced in some patients, (b) Lymphocyte proliferation and NK cell activity which were suppressed in cancer patients can be significantly restored by Naturin treatment, (c) the addition of Naturin treatment to patients receiving radiotherapy and chemotherapy augments immune response and reduces radiation and chemotherapy injury, and (d) no cytotoxic side effects were found in patients given Naturin treatment for up to eight months.

Staurosporine inhibits interferon alpha-induced gene expression in Friend erythroleukemia cells through a PKC independent pathway.

Interferons (IFNs) are able to induce an increased transcription of several genes, which can occur within minutes of the binding of IFNs to their receptors. The specific induced transcription is mediated by the interaction of specific transcription factors with regulatory DNA sequences that lie upstream the promoters of IFN induced genes. Phosphorylation of IFN-specific transcription factors is required for activation of transcription. We have studied the antiviral effect and the induction of gene expression by IFN-alpha in Friend Leukemia cells (FLC) in the presence of a series of inhibitors of known kinases. Protein kinase C (PKC)-specific inhibitors, i.e. calphostin C and bisindolylmaleimide, failed to influence the IFN-induced gene expression and the antiviral state. Likewise, little or no effect was found using inhibitors such as H7 or K252a. Chronic exposure of FLC to phorbol ester, that causes down regulation of PKC (the effectiveness of TPA treatment was proven by PKC enzymatic assay), has no effect on IFN-alpha action. In addition, treatment of FLC with staurosporine prevented the induction of IFN-stimulated genes and the establishment of the antiviral state only when this drug was used at high dosage (500 nM). This result indicates that, also in FLC, activation of PKC is not involved in the transcriptional response of the cells to IFN-alpha treatment. The non receptor tyrosine kinases of the JAK family that take part in the IFNs-specific transduction pathways could be the target of the staurosporine specific inhibition of the IFN-alpha action.

Glycosylation of glycoprotein 55 encoded by the anaemia-inducing strain of Friend spleen focus-forming virus.

Normal rat kidney cells, non-productively infected with the anaemia-inducing variant of Friend spleen focus-forming virus (F-SFFVA), were metabolically labelled with [2-3H]mannose. The primary translation product of the viral envelope gene (env), representing a glycoprotein with an apparent molecular M(r) of 55,000 (gp55), was isolated from cell lysates by immunoaffinity chromatography and purified by preparative SDS/PAGE. Radiolabelled oligosaccharides, released from tryptic glycopeptides by treatment with endo-beta-N-acetylglucosaminidase H, were characterized chromatographically, by enzymic digestion and by acetolysis. The results revealed that F-SFFVA gp55 obtained from this source carried predominantly oligomannose type sugar chains with five to nine mannoses. As a characteristic feature, glycans with seven to nine mannoses contained, in part, an additional glucose residue. Although the amount of glucosylated species found was higher in F-SFFVA gp55 (about 25% of total endo-H-sensitive oligosaccharides) than in gp55 of the corresponding polycythaemia-inducing variant (F-SFFVP, 16.3%), the overall glycosylation pattern of the F-SFFVA env product closely resembled that of F-SFFVP gp55 [Strube et al. (1988) J Biol Chem 263:3762-71]. Hence, our results demonstrate that the different intracellular processing and transport of the primary F-SFFVA env product cannot be attributed to aberrant trimming of its oligomannose type glycans.

Efficacy of recombinant human macrophage colony-stimulating factor in combination with whole-body hyperthermia in the treatment of mice infected with the polycythemia-inducing strain of the Friend virus complex.

Macrophage colony-stimulating factor (M-CSF, CSF-1) and whole-body hyperthermia (WBH) were evaluated, alone or in combination, for their capability to influence disease progression in mice inoculated with the polycythemia-inducing strain of the Friend virus complex (FVC-P). DBA/2 mice were injected i.v. with FVC-P and were treated with 20 micrograms/dose M-CSF s.c. twice a day for 5 days beginning 6 days after injection of FVC-P and/or with WBH (between 38.8 degrees C and 40.2 degrees C) given on days 5 and 12 after FVC-P injection. Fourteen days after viral inoculation, mice were sacrificed and spleen cells evaluated for: 1) spleen focus-forming virus (SFFV), by the spleen focus-forming unit assay (SFFU); 2) SFFV mRNA and genomic DNA using, respectively, Northern and Southern analysis with a B-E-SFFV DNA probe; and 3) natural killer (NK) cell activity, by 51Cr-release assay. Treatment with M-CSF or WBH alone had a small effect on SFFU numbers but little or no effect on SFFV mRNA expression and SFFV-specific DNA. However, dramatically decreased levels of SFFU and SFFV mRNA and specific DNA fragments were observed in mice treated with M-CSF in combination with WBH, and NK cell activity was restored to normal. These results suggest the possibility that M-CSF may have a therapeutic effect in combination with WBH in the in vivo treatment of certain hematologic malignancies and/or retroviral infections.

A study of the mechanism of selenite-induced hypomethylated DNA and differentiation of Friend erythroleukemic cells.

Sodium selenite is a good inducer of hemoglobin production in Friend erythroleukemic cells (FELC). At a concentration of 20 microM 70-80% of the cells produce hemoglobin and the DNA is hypomethylated. What is the mechanism for sodium selenite alteration of the DNA methylation pattern? Experiments with methionine adenosyltransferase (the enzyme that synthesizes adenosylmethionine) showed little effect of selenite on the activity of this enzyme in vitro or in vivo. Therefore, FELC are able to synthesize S-adenosylmethionine in the presence of sodium selenite. When sodium selenite was added to an in vitro assay for DNA methylase, the enzyme was non-competitively inhibited by 80% at 20 microM selenite with a Ki of 6 microM. DNA methylase isolated from control and selenite-treated FELC was purified through a DEAE-Sephacel column and no difference in activity was found. In the presence of selenite, DNA methylase is very sensitive to selenite inhibition, but removal of the selenite restores activity. However, DNA synthesized by FELC grown in the presence of selenite (no DNA methylase activity) was found to be hypomethylated. These results suggest that DNA methylase activity is inhibited in FELC grown in the presence of sodium selenite.

Prostaglandin A1 induces differentiation in Friend erythroleukemia cells.

The effect of different prostaglandins and prostaglandin-metabolites on the growth and differentiation of Friend erythroleukemia cells (FLC) was evaluated. The prostaglandin-metabolites, thromboxane B2 and 6-keto PGF1 alpha, were completely inactive, while PGE1 inhibited slightly and PGF2 alpha stimulated the replication of FLC. PGA1 was found to be the most active compound. It profoundly inhibited the replication of both DMSO-treated and undifferentiated FLC. Most importantly, PGA1 alone induced differentiation in FLC, stimulating hemoglobin production over a five-day period. PGA1-stimulated differentiation was completely suppressed by the addition of 10(-6)M hydrocortisone. Finally, treatment of DMSO-differentiated cells with PGA1 (but no DMSO) prevented the return to the undifferentiated state.

XC cell fusion by murine leukemia viruses: fusion from without.

Concentrated murine leukemia virus (MuLV) or MuLV producing cells induce XC cell fusion within an hour leading to syncytia formation. While MuLV inactivated by UV irradiation, beta-propiolactone or hydroxylamine treatment still caused cell fusion, Bromelin- or trypsin treated MuLV was no longer able to fuse XC cells. Though sonicated MuLV induced no XC cell fusion, it interfered with cell fusion as caused by untreated MuLV. XC cells infected by diluted MuLV of a titer lower than 1 X 10(5) PFU/ml formed no syncytia although they produced MuLV. The cell fusion mechanism is discussed.

Antineoplastic activity of cannabinoids.

Lewis lung adenocarcinoma growth was retarded by the oral administration of delta9-tetrahydrocannabinol (delta9-THC), delta8-tetrahydrocannabinol (delta8-THC), and cannabinol (CBN), but not cannabidiol (CBD). Animals treated for 10 consecutive days with delta9-THC, beginning the day after tumor implantation, demonstrated a dose-dependent action of retarded tumor growth. Mice treated for 20 consecutive days with delta8-THC and CBN had reduced primary tumor size. CBD showed no inhibitory effect on tumor growth at 14, 21, or 28 days. Delta9-THC, delta8-THC, and CBN increased the mean survival time (36% at 100 mg/kg, 25% at 200 mg/kg, and 27% at 50 mg/kg, respectively), whereas CBD did not. Delta9-THC administered orally daily until death in doses of 50, 100, or 200 mg/kg did not increase the life-spans of (C57BL/6 times DBA/2)F1 (BDF1) mice hosting the L1210 murine leukemia. However, delta9-THC administered daily for 10 days significantly inhibited Friend leukemia virus-induced splenomegaly by 71% at 200 mg/kg as compared to 90.2% for actinomycin D. Experiments with bone marrow and isolated Lewis lung cells incubated in vitro with delta9-THC and delta8-THC showed a dose-dependent (10(-4)-10(-7)) inhibition (80-20%, respectively) of tritiated thymidine and 14C-uridine uptake into these cells. CBD was active only in high concentrations (10(-4)).