Small-molecule targeting of MUSASHI RNA-binding activity in acute myeloid leukemia.

The MUSASHI (MSI) family of RNA binding proteins (MSI1 and MSI2) contribute to a wide spectrum of cancers including acute myeloid leukemia. We find that the small molecule Ro 08-2750 (Ro) binds directly and selectively to MSI2 and competes for its RNA binding in biochemical assays. Ro treatment in mouse and human myeloid leukemia cells results in an increase in differentiation and apoptosis, inhibition of known MSI-targets, and a shared global gene expression signature similar to shRNA depletion of MSI2. Ro demonstrates in vivo inhibition of c-MYC and reduces disease burden in a murine AML leukemia model. Thus, we identify a small molecule that targets MSI's oncogenic activity. Our study provides a framework for targeting RNA binding proteins in cancer.

Phenethyl isothiocyanate promotes immune responses in normal BALB/c mice, inhibits murine leukemia WEHI-3 cells, and stimulates immunomodulations in vivo.

Enhanced cruciferous vegetable consumption is associated with the reduction of cancer incidence as shown in epidemiological studies. Phenethyl isothiocyanate (PEITC), one of the important compounds in cruciferous vegetables, has been shown to induce apoptosis in many types of human cancer cell lines, but there is no available information addressing the effects on normal and leukemia mice in vivo. The purpose of this study is to focus on the in vivo effects of PEITC on immune responses of normal and WEHI-3 leukemia BALB/c mice in vivo. Influences of PEITC on BALB/c mice after intraperitoneal (i.p.) injection with WEHI-3 cells and normal mice were investigated. In normal BALB/c mice, PEITC did not affect the body weight when compared to the olive oil treated animals. Moreover, PEITC promoted phagocytosis by macrophages from peripheral blood mononuclear cells (PBMC) and peritoneal cavity, increased the levels of CD11b and Mac-3, decreased the level of CD19 and promoted natural killer (NK) cell cytotoxic activity, but it did not alter the level of CD3. Also, PEITC enhanced T cell proliferation after concanavalin A (Con A) stimulation. Otherwise, PEITC increased the body weight, but decreased the weight of liver and spleen as compared to the olive oil-treated WEHI-3 leukemia mice. PEITC also increased the level of CD19, decreased the levels of CD3 and Mac-3 rather than influence in the level of CD11b, suggesting that the differentiation of the precursor of macrophages and T cells was inhibited, but the differentiation of the precursor of B cells was promoted in leukemia mice. Furthermore, PEITC enhanced phagocytosis by monocytes and macrophages from PBMC and peritoneal cavity, and also promoted the NK cell cytotoxic activity in comparison with the group of leukemia mice. Based on these observations, the biological properties of PEITC can promote immune responses in normal and WEHI-3 leukemia mice in vivo. © 2011 Wiley Periodicals, Inc. Environ Toxicol, 2013.

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.

Blood transfusion promotes cancer progression: a critical role for aged erythrocytes.

BACKGROUND: In cancer patients, allogeneic blood transfusion is associated with poorer prognosis, but the independent effect of the transfusion is controversial. Moreover, mediating mechanisms underlying the alleged cancer-promoting effects of blood transfusion are unknown, including the involvement of donors' leukocytes, erythrocytes, and soluble factors. METHOD: Two syngeneic tumor models were used in Fischer 344 rats, the MADB106 mammary adenocarcinoma and the CRNK-16 leukemia. Outcomes included host ability to clear circulating cancer cells, and host survival rates. The independent impact of blood transfusion was assessed, and potential deleterious characteristics of the transfusion were studied, including blood storage duration; the role of erythrocytes, leukocyte, and soluble factors; and the kinetics of the effects. RESULTS: Blood transfusion was found to be an independent and significant risk factor for cancer progression in both models, causing up to a fourfold increase in lung tumor retention and doubling mortality rates. Blood storage time was the critical determinant of these deleterious effects, regardless of whether the transfused blood was allogeneic or autogenic. Surprisingly, aged erythrocytes (9 days and older), rather than leukocytes or soluble factors, mediated the effects, which occurred in both operated and nonoperated animals. The effects of erythrocytes transfusion in the MADB106 model emerged immediately and dissipated within 24 h. CONCLUSIONS: In rats, transfusion of fresh blood is less harmful than transfusion of stored blood in the context of progressing malignancies. Further studies should address mediating mechanisms through which erythrocytes' storage duration can impact the rate of complications while treating malignant diseases and potentially other pathologies.

Evidence for an association of high levels of endogenous Acetyl-Ser-Asp-Lys-Pro, a potent mediator of angiogenesis, with acute myeloid leukemia development.

Evidence from clinical and laboratory studies suggests that angiogenesis is important in the progression of solid tumours and hematologic malignancies. We have shown that the naturally occurring tetrapeptide Acetyl-Ser-Asp-Lys-Pro (AcSDKP) is a potent angiogenic factor normally present at nanomolar concentrations in the blood. A murine leukemia model was used to assess whether there was a correlation between levels of endogenous AcSDKP and the development of disease. Levels of AcSDKP in the plasma and bone marrow (BM) cells from mice bearing an acute myeloid leukemia (AML) were five- to ten-fold greater than those in non-leukemic mice. Furthermore, a strong correlation between the concentration of endogenous AcSDKP and the progression of AML was demonstrated. These results are consistent with the marked increase in BM vascularity observed in leukemic mice. The physiologic relevance of these findings awaits further studies and the contribution of AcSDKP to the pathogenesis of leukemia is under investigation.

Cooperative antitumor effects of vitamin D3 derivatives and rosemary preparations in a mouse model of myeloid leukemia.

1alpha,25-dihydroxyvitamin D(3) (1,25D(3)) is a powerful differentiation agent, which has potential for treatment of myeloid leukemias and other types of cancer, but the calcemia produced by pharmacologically active doses precludes the use of this agent in the clinic. We have shown that carnosic acid, the major rosemary polyphenol, enhances the differentiating and antiproliferative effects of low concentrations of 1,25D(3) in human myeloid leukemia cell lines (HL60, U937). Here we translated these findings to in vivo conditions using a syngeneic mouse leukemia tumor model. To this end, we first demonstrated that as in HL60 cells, differentiation of WEHI-3B D(-) murine myelomonocytic leukemia cells induced by 1 nM 1,25D(3) or its low-calcemic analog, 1,25-dihydroxy-16-ene-5,6-trans-cholecalciferol (Ro25-4020), can be synergistically potentiated by carnosic acid (10 microM) or the carnosic acid-rich ethanolic extract of rosemary leaves. This effect was accompanied by cell cycle arrest in G0 + G1 phase and a marked inhibition of cell growth. In the in vivo studies, i.p. injections of 2 microg Ro25-4020 in Balb/c mice bearing WEHI-3B D(-) tumors produced a significant delay in tumor appearance and reduction in tumor size, without significant toxicity. Another analog, 1,25-dihydroxy-16,23Z-diene-20-epi-26,27-hexafluoro-19-nor-cholecalciferol (Ro26-3884) administered at the same dose was less effective than Ro25-4020 and profoundly toxic. Importantly, combined treatment with 1% dry rosemary extract (mixed with food) and 1 microg Ro25-4020 resulted in a strong cooperative antitumor effect, without inducing hypercalcemia. These results indicate for the first time that a plant polyphenolic preparation and a vitamin D derivative can cooperate not only in inducing leukemia cell differentiation in vitro, but also in the antileukemic activity in vivo. These data may suggest novel protocols for chemoprevention or differentiation therapy of myeloid leukemia.

Establishment of a transplantable human myeloid BALB/c nude mouse model.

To establish a mouse model bearing transplantable human chronic myeloid leukemia for hematopoietic stem cell transplantation to treat leukemia, 4 - 5-week-old female BALB/c nude mice were given cyclophosphamide 2 mg/mouse at day -2, -1, and then the human chronic myeloid leukemia K562 cells were engrafted into the mice at day 0 by injection via tail vein or peritoneal cavity. PB and BM cells were collected, the CD45, CD13, and CD33 antigens were delected by using FCM, the bcr/abl fusion gene mRNA was examined by RT-PCR. The results showed that transplantable leukemic mice could be yielded from 4 - 5-week-old nude mice either by injection through tail vein or peritoneal cavity when the total number of inoculated tumor cells was more than 2 x 10(5) per mouse, whether being pretreated with 2 mg CTX/mouse or not. The transplanted mice could survive 30 - 60 day with leukemia. In conclusion, the mouse model bearing leukemia can be established by inoculation 2 x 10(5) K562 cells into immunodeficient BALB/c nude mice.

[Establishment and characterization of a human acute monocytic leukemic cell line, SHI-1, carrying t(6;11)(q27;23) and p53 gene alteration].

OBJECTIVE: To establish a novel human monocytic leukemic cell line and characterize its biological features. METHODS: Mononuclear cells isolated from the bone marrow of an acute monocytic leukemia (AML-M(5b)) patient at relapse were inoculated in a liquid culture system. And the biologic features of the established cell line SHI-1 were characterized by morphology, cytochemical staining, flow cytometry, karyotypic analysis, polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR), fluorescence in situ hybridization (FISH), tumorigenicity in nude mice, quantitative fluorescent polymerase chain reaction, broth medium culture, short tandem repeating sequences-PCR (STR-PCR), multiplex-FISH (M-FISH), and (3)H-thymidine incorporation assay. RESULTS: A human acute monocytic leukemia cell line, SHI-1, was established and has proliferated continuously in vitro for over one year. The cell line presented typical morphology and immuno-profile of monocytic lineage with the original t(6;11)(q27;q23) and del(17)(p11) abnormalities. The MLL-AF6 fusion transcript was detected by RT-PCR. The rearrangement of MLL gene, deletion of p53 gene, and translocation between chromosomes 6 and 11 were revealed by FISH. A point mutation of ATC-->ACC at exon 6 of the p53 gene was found by sequencing of the PCR products. The clonality and the high tumorigenicity of the SHI-1 cell line were confirmed. Infections of EBV and mycoplasma were excluded. A derivative chromosome 7 resulting from a translocation between chromosomes 7 and 13, monosomy 18 and a minute derived from chromosome 8 in addition to t(6;11) and deletion(17)(p11) were detected by M-FISH in SHI-1 cells passaged to March 2003. Cell line authentication by STR-PCR confirmed the identity to the original leukemic cells of the patient. (3)H-thymidine incorporation assay showed that IL-4 and IL-15 had proliferative effects, while IFN-gamma, TNFalpha, IL-2, PDGF, and IL-7 had inhibitory effects on the cell line. CONCLUSIONS: SHI-1 is a novel acute monocytic leukemia-derived cell line carrying t(6;11)(q27;q23) and p53 gene alteration and having high tumorigenicity in nude mice. It provides a new useful tool for leukemia research.

[Spontaneous leukosis as a model for an investigation of low and very low physical and physico-chemical effects on oncological process]. / Spontannyi leikoz -- model' dlia izucheniia éffektov malykh i sverkhmalykh doz fizicheskikh i fiziko-khimicheskikh vozdeistvii na opukholevyi protsess.

Kinetic investigation of spontaneous leukosis in AKR mice have been carried in connection with a number of indices: changes in the mass of principal organs of the immune systems (thymus, spleen, lymphatic nodes), liver, alterations of haematological data (the sum of leukocytes, the percentage composition of blood cells, the quantity of undifferentiated cells), changes of physico-chemical conditions in cells (NMR-investigation). The dynamics of some of these indices and also life-spans of animals with leukosis after irradiation with doses 1.2-2.4 cGy (dose-rate 0.6 cGy/day) have been investigated. The enhancement of the frequency of leukosis and shortening the average and maximum life-spans of irradiated mice has been found.

Suppression of retrovirus-induced immunodeficiency disease (murine AIDS) by trimidox and didox: novel ribonucleotide reductase inhibitors with less bone marrow toxicity than hydroxyurea.

Recently, the use of the ribonucleotide reductase (RR) inhibitor hydroxyurea (HU) in combination with nucleoside analogs has gained attention as a potential strategy for anti-HIV-1 therapy. However, appeal for the long-term use of HU in HIV-1 infection may be limited by its propensity to induce hematopoietic toxicity. We report a comparison of the efficacy and bone marrow toxicity of HU (400 and 200 mg/kg/day) with the novel RR inhibitors and free radical-scavenging compounds didox (DX; 3,4-dihydroxybenzohydroxamic acid; 350 mg/kg/day) and trimidox (TX; 3,4,5-trihydroxybenzamidoxime; 175 mg/kg/day) in the murine AIDS (LPBM5 MuLV) model of retrovirus infection. Infected mice received daily drug treatment for 8 weeks. Efficacy was determined by measuring drug effects on retroviral-induced disease progression (i.e. development of splenomegaly and hypergammaglobulinemia) and by evaluating splenic levels of proviral DNA. Bone marrow toxicity was evaluated by measuring peripheral blood indices (WBC, hematocrit and reticulocyte counts), femoral cellularity and by determining the numbers of hematopoietic progenitor cells (CFU-GM, BFU-E) per femur and spleen. Compared to infected controls receiving no drug treatment, disease progression was significantly suppressed by TX, DX and HU. However, HU was associated with mortality and induced significant hematopoietic toxicity in a time- and dose-dependent manner. Conversely, TX and DX effectively inhibited retrovirus-induced disease but did not induce hematopoietic toxicity. These results suggest that due to their reduced hematopoietic toxicity and ability to inhibit disease progression in murine AIDS, TX and DX may offer effective alternatives to HU therapy in HIV-1 infection.

Gender-related differences in susceptibility, early virus dissemination and immunosuppression in mice infected with Friend murine leukaemia virus variant FIS-2.

An emerging amount of data indicates a correlation between gender-related factors and regulation of virus infection and supports what is known in clinical circles, that these topics are of great importance in many infectious diseases. In the present study we found that young adult NMRI male mice are more susceptible to infection by a variant of Friend murine leukaemia virus, FIS-2, than are female mice. We observed that the level of virus in serum, bone marrow and spleen was initially higher in male mice. Male mice were also more susceptible to FIS-2-induced immunosuppression. These results indicate a more efficient virus replication and dissemination in male mice. Studies with recombinant viruses between FIS-2 and the prototype Friend murine leukaemia virus revealed that FIS-2 LTR is one major factor contributing to the observed gender differences. A possible sex hormone influence on FIS-2 transcription due to the presence of a glucocorticoid response element in FIS-2 LTR is discussed.

[Dynamic of DNAse activity induction in serum of normal animals after heterogeneous DNA injection and in hereditary pathology]. / Dinamika induktsii DNKaznoi aktivnosti v syvorotke krovi zhivotnykh pri vvedenii geterogennoi DNK v norme i pri nasleduemoi patologii.

Studies of reactions in animals with hereditary diseases (Sapphire minks highly sensitive to Aleutian disease virus, ADV; CBA mice with 60-70% incidence of tumors; AKR mice with 90% incidence of leukemia) showed that serum DNAse activity in these animals dropped after injection of a foreign heterogeneous DNA and remained decreased during 72 h. By contrast, serum DNAse activity considerably and persistently increased after injection of DNA in Standard minks resistant to ADV, C57BI/6J mice with 1% tumor incidence, and random-bred albino mice. Presumably the capacity of standard minks to react to a foreign heterogeneous DNA by increase of DNAse activity ensures their resistance to DNA-containing ADV, while incapacity of Sapphire minks to respond to DNA by DNAse activity induction makes them sensitive to ADV. A similar relationship between the capacity to react to DNA by changes in serum DNAse activity and capacity to inherit a disease was detected in mouse strains.

Facilitation of functional compartmentalization of bone marrow cells in leukemic mice by biological response modifiers: an immunotherapeutic approach.

Biological Response Modifiers (BRMs) including interleukin-2 (IL-2), interferon-gamma (IFN-gamma) and sheep erythrocytes (SRBC) protected N,N'-ethylnitrosourea (ENU) induced leukaemic mice. Two cell types from the bone marrow were isolated in density specific gradient representing two distinct compartments, the low density cells being more CD34 positive than the high density group. Investigations with the functional efficacy of such compartments revealed significant improvement of cytotoxic efficacy and phagocytic burst at the high density compartment (HDC) level. The high density compartment was found to be more responsive towards the BRMs compared to the cells of the low density compartment (LDC). It was suggested that use of BRMs in vivo can stimulate a potent functional progenitor compartmentalization in normal as well as leukaemic mice. These observations are expected to help a logistic approach towards combined BRM therapy at the clinical level.

Absence of the effects of 50 Hz magnetic fields on the progression of acute myeloid leukaemia in rats.

PURPOSE: As the most recent epidemiological studies provide no definite conclusions about the effects of 50/60 Hz magnetic fields (MFs) on the incidence of leukaemia in humans, animal models in a well-controlled environment are useful for evaluating the possibility of an association between MFs and leukaemia. The present study was designed to determine whether 50 Hz magnetic fields can alter the progression of leukaemia. MATERIALS AND METHODS: A well-characterized model of transplantable acute myeloid leukaemia in rats was used for the first time. This model is closely related to human acute myeloid leukaemia, the type most frequently reported in epidemiological studies of adults. After leukaemic cell implantation, rats were exposed to a sinusoidal 50 Hz MF of 100 microT for 18 h a day, 7 days a week, throughout leukaemia progression. The parameters investigated were: survival time, body weight, haematologic parameters, infiltration of blood, bone marrow, spleen and liver by leukaemic cells. RESULTS: The results showed no significant changes (p > 0.05) in leukaemic MF-exposed versus unexposed rats for any of the parameters involved in leukaemia progression. CONCLUSION: These data do not support the hypothesis that 50 Hz magnetic fields influence leukaemia progression in humans.

Alteration of cytokine levels in murine retrovirus infection: modulation by combination therapy.

Immunoregulatory cytokines may regulate the resistance or susceptibility of a host to retroviral infection. These cytokines may be therapeutically modulated to prevent or limit the progression of infection. The non-progression to AIDS of some HIV+ patients has been related to a strong type 1 cytokine response (IL-2, IL-12, and IFNgamma). For this reason, we investigated the ability of combination therapeutics to modulate cytokines in vivo towards a type 1 cytokine response in a murine retroviral infection using Friend leukemia virus (FLV). BALB/c mice were infected with FLV and treated with either 3'-azido-3'-deoxythymidine (AZT), the immunomodulator methionine enkephalin (MENK), or a combination of both AZT and MENK starting 3 d post infection. Splenocytes were harvested on days 1, 3, 7, 14, 21 and 28 post treatment initiation and cultured with 1 microg/ml concanavalin A (ConA) for 24 h. Supernatants were examined for IL-2, IL-4, IL-10, IL-12, and IFNgamma cytokine production using cytokine specific ELISAs. The levels of type 2 cytokines were not significantly changed by any treatment group over the course of the disease. However, although decreased in all infected animals, type 1 cytokines were partially maintained by the combination treatment through day 21. RT-PCR for cytokine specific mRNA confirmed these results, with expression of the type 1 cytokines, especially IFNgamma, being maintained through day 21. Establishment of a treatment regime that can maintain protective cytokine activities against disease progression may prove applicable to other retroviral infections.

Identification of directly infected cells in the bone marrow of neonatal moloney murine leukemia virus-infected mice by use of a moloney murine leukemia virus-based vector.

Early bone marrow infection of Moloney murine leukemia virus (M-MuLV)-infected mice was studied. Previous experiments indicated that early bone marrow infection is essential for the efficient development of T lymphoma. In order to identify the cellular pathway of infection in the bone marrow, infection of mice with a helper-free replication-defective M-MuLV-based retroviral vector was carried out. Such a vector will undergo only one round of infection, without spreading to other cells; thus, cells infected by the initially injected virus (directly infected cells) can be identified. For these experiments, the BAG vector that expresses bacterial beta-galactosidase was employed. Neonatal NIH/Swiss mice were inoculated intraperitoneally with ca. 10(6) infectious units of a BAG vector pseudotyped with M-MuLV proteins, and bone marrow cells were recovered 2 to 12 days postinfection. Single-cell suspensions were tested for infection by staining with X-Gal (5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside) or by immunofluorescence with an anti-beta-galactosidase antibody. Two sizes of infected cells were evident: large multinucleated cells and small nondescript (presumptively hematopoietic) cells. Secondary stains for lineage-specific markers indicated that the large cells were osteoclasts. Some of the small cells expressed nonspecific esterase, which placed them in the myeloid lineage, but they lacked markers for hematopoietic progenitors (mac-1, gr-1, sca-1, and CD34). These results provide evidence for primary M-MuLV infection of osteoclasts or osteoclast progenitors in the bone marrow, and they suggest that known hematopoietic progenitors are not primary targets for infection. However, the subsequent spread of infection to hematopoietic progenitors was indicated, since bone marrow from mice infected in parallel with replication-competent wild-type M-MuLV showed detectable infection in small cells positive for mac-1 or CD34, as well as in osteoclasts.

G-CSF mobilization of haemopoietic cell populations in SCID mice engrafted with human leukaemia.

We have studied granulocyte colony-stimulating factor (G-CSF)-induced mobilization of haemopoietic cells in severe combined immune-deficient (SCID) mice engrafted with human leukaemia. Three leukaemia cell lines were investigated: the HL60 myeloblastic cell line, a chronic myeloid leukaemia (CML) xenograft cell line and an acute myeloid leukaemia (AML) xenograft line. Engraftment was detected using immunofluorescent staining of class I human leukocyte antigens and flow cytometry. All the tumours grew as disseminated disease with engraftment of bone marrow preceding involvement of peripheral blood (PB). After treatment with G-CSF (250 microg/kg/day) for 5 days, mobilization of haemopoietic progenitor cells (HPCs) was observed in non-engrafted SCID mice (40-fold) and in mice engrafted with human leukaemia (20-fold). G-CSF stimulated increases in PB HPCs and total numbers of host nucleated cells in leukaemia-bearing mice but did not induce rises in numbers of circulating HL60 colony-forming cells. Similarly, in mice engrafted with human CML or AML, G-CSF did not increase the number of malignant cells in the PB. These results provided evidence that the migration of normal and malignant haemopoietic cells into the PB are controlled by different mechanisms, and that contamination of PBSC harvests with leukaemic cells in SCID-human chimaeric mice is not enhanced by G-CSF-stimulated mobilization.

Preclinical pharmacology of ecteinascidin 729, a marine natural product with potent antitumor activity.

Ecteinascidins are marine natural products with potent antiproliferative activity under evaluation as chemotherapeutic agents by the National Cancer Institute. Ecteinascidins bind the minor groove of DNA and may form covalent adducts with DNA by binding the N-2 of guanine in a fashion similar to saframycin antibiotics. The most potent ecteinascidin is ET-729 with antitumor activity observed following administration of 3.8 and 10 micrograms/kg to mice bearing P388 leukemia and B16 melanoma, respectively. A reverse-phase high-performance liquid chromatography (HPLC) assay and an L1210 cell bioassay were developed for ET-729 and utilized for stability and murine pharmacokinetic studies. HPLC analysis showed that ET-729 was stable in organic solvents, mobile phase and acidic buffer (t1/2 > 100 h). Stability was diminished under neutral and basic conditions (t1/2 < 14 h). Following a 48-h incubation with L1210 cells in growth medium in the absence and presence of 2.5% murine plasma, the 50% growth inhibitory concentrations (IC50) of ET-729 were 37 and 72 pM, respectively. Following intravenous administration of ET-729 to male CD2F1 mice, the disappearance of antiproliferative activity determined by the bioassay was described by a two-compartment open model. The mean values of the elimination half-life and plasma clearance were 28 min and 39.7 ml/min per kg, respectively. Following intraperitoneal administration, peak plasma concentrations of antiproliferative activity were observed 6-15 min after injection and antiproliferative concentrations remained above 1 nM for longer than 1 h. Intraperitoneal bioavailability varied over a wide range (20-91%). Antiproliferative activity was detected in every urine sample following intravenous and intraperitoneal administration, but the total 48-h urinary recovery was less than 0.1%.

10A1 MuLV induces a murine leukemia that expresses hematopoietic stem cell markers by a mechanism that includes fli-1 integration.

The 10A1 murine leukemia virus induces tumors that lack lineage-specific markers found on myeloid, T-cell, and B-cell lineages. Either erythroid or multipotent stem cells can have this phenotype; therefore we have used fluorescence-activated cell sorter analysis with either multipotent stem cell markers or markers found on lineage-restricted precursors to differentiate between these two possibilities. The results showed that tumors induced by 10A1 expressed multipotent stem cell markers as well as some lineage-restricted precursor markers. To further study the tumor phenotype, we analyzed total RNAs from 10A1-induced tumors by Northern blotting for c-kit, erythropoietin receptor, and T-cell gamma receptor mRNAs. Most of the tumors contained these mRNAs, which are characteristic of early hematopoietic cells. These results are consistent with the hypothesis that 10A1-induced tumor cells are early multipotent hematopoietic stem cells. Southern blot analysis revealed that 14 of 14 10A1-induced tumor cell DNAs examined contained MuLV integrations into the fli-1 gene. The results strongly suggested that promoter insertion into fli-1 is required for tumor formation.

Melatonin concentrations in pineal organ culture are suppressed by sera from tumor-bearing mice.

The melatonin rhythm is significantly attenuated in a wide range of human and animal tumor types. Since surgical removal of the tumor has been shown to restore this rhythm, we hypothesized that a plasma borne tumor-associated factor (TAMF) could be responsible. A population of mice were injected with tumor cells and sequentially killed and bled over the following 9 days, i.e., to the maximal state of tumor growth. Pooled serum from the different collection days was added to an established pineal organ culture system, and melatonin concentrations measured. A highly significant correlation between melatonin concentrations and the stage of tumor growth was seen with maximal inhibition occurring at day 9 (P < 0.01). These findings support our hypothesis and may help to explain the mechanism whereby melatonin rhythmicity is suppressed in cancer patients.