Antitumor effects of citrinin in an animal model of Sarcoma 180 via cytogenetic mechanisms.

Citrinin (CIT) is a cytotoxic, hepatotoxic, nephrotoxic and cardiotoxic metabolite obtained from Penicillium citrinum, that has been increasingly searched as an anticancer drug candidate. In this study, we assessed the antitumor effects of citrinin, using cytogenetic biomarkers for genotoxicity in Sarcoma 180 (S-180) ascitic fluid cells of mice. Citrinin, extracted from P. citrinum acetonitrile extract, was characterized by LC-MS. Cytotoxic assessment was done through using comet (alkaline version) and micronucleus assays. In S-180 cells, CI50 of CIT was 3.77 µg/mL, while at 12.5 and 100 µg/mL, CIT was as cytotoxic as doxorubicin (2 µg/mL). At 0.5, 1.0 and 2.0 µg/mL, it induced genotoxicity and mutagenicity in S-180 cells, especially at 2 µg/mL, triggering oxidative damage similar to hydrogen peroxide (10 mM). The antitumor effects were evidenced by a marked increase in S-180 cells apoptosis and necrosis due to clastogenic and/or aneugenic cytogenetic effects (micronucleus formation), as well as by induction of nucleoplasm bridges and nuclear buds, culminating in S-180 apoptosis and necrosis. CIT has potential as drug candidate for antitumor purposesbyinvolving cytogenetic mechanisms.

Construction of random tumor transcriptome expression library for creating and selecting novel tumor antigens.

Novel tumor antigens are necessary for the development of efficient tumor vaccines for overcoming the immunotolerance and immunosuppression induced by tumors. Here, we developed a novel strategy to create tumor antigens by construction of random tumor transcriptome expression library (RTTEL). The complementary DNA (cDNA) from S180 sarcoma was used as template for arbitrarily amplifying gene fragments with random primers by PCR, then ligated to the C-terminal of HSP65 in a plasmid pET28a-HSP for constructing RTTEL in Escherichia coli. A novel antigen of A5 was selected from RTTEL with the strongest immunotherapeutic effects on S180 sarcoma. Adoptive immunotherapy with anti-A5 sera also inhibited tumor growth, further confirming the key antitumor roles of A5-specific antibodies in mice. A5 contains a sequence similar to protein-L-isoaspartate (D-aspartate) O-methyltransferase (PCMT1). The antisera of A5 were verified to cross-react with PCMT1 by Western blotting assay and vice versa. Both anti-A5 sera and anti-PCMT1 sera could induce antibody-dependent cell-mediated cytotoxicity and complement-dependent cytotoxicity toward S180 cells by in vitro assay. Further assay with fluorescent staining showed that PCMT1 is detectable on the surface of S180 cells. Summary, the strategy to construct RTTEL is potential for creating and screening novel tumor antigens to develop efficient tumor vaccines. By RTTEL, we successfully created a protein antigen of A5 with significant immunotherapeutic effects on S180 sarcoma by induction of antibodies targeting for PCMT1.

A selenosemicarbazone complex with copper efficiently down-regulates the 90-kDa heat shock protein HSP90AA1 and its client proteins in cancer cells.

BACKGROUND: The 90-kDa heat shock protein HSP90AA1 is critical for the stability of several proteins that are important for tumor progression and thus, is a promising target for cancer therapy. Selenosemicarbazone metal complexes have been shown to possess anticancer activity through an unknown molecular mechanism. METHODS: The MTT assay, fluorescence-activated cell sorting, and fluorescent microscopy were used to analyze the mechanism of the anti-cancer activity of the selenosemicarbazone metal complexes. Additionally, RNA-seq was applied to identify transcriptional gene changes, and in turn, the signaling pathways involved in the process of 2-24a/Cu-induced cell death. Last, the expression of HSP90AA1, HSPA1A, PIM1, and AKT proteins in 2-24a/Cu-treated cells were investigated by western blot analysis. RESULTS: A novel selenosemicarbazone copper complex (2-24a/Cu) efficiently induced G2/M arrest and was cytotoxic in cancer cells. 2-24a/Cu significantly induced oxidative stress in cancer cells. Interestingly, although RNA-seq revealed that the transcription of HSP90AA1 was increased in 2-24a/Cu-treated cells, western blotting showed that the expression of HSP90AA1 protein was significantly decreased in these cells. Furthermore, down-regulation of HSP90AA1 led to the degradation of its client proteins (PIM1 and AKT1), which are also cancer therapy targets. CONCLUSION: Our results showed that 2-24a/Cu efficiently generates oxidative stress and down-regulates HSP90AA1 and its client proteins (PIM1, AKT1) in U2os and HeLa cells. These results demonstrate the potential application of this novel copper complex in cancer therapy.

Administration of vitamin E prevents thymocyte apoptosis in murine sarcoma S180 tumor bearing mice.

Many reports claimed that thymic involution occurred in tumor bearing host, which led to a reduction of proliferation in T cells and an impaired immune system. In this study, murine sarcoma S180 tumor cells were used to establish tumor model. There was distinct apoptosis in thymus of the S180 tumor bearing host after 3-week tumor inoculation. High level reactive oxygen species generation was detected in thymocytes of S180 tumor bearers. Vitamin E, a potent antioxidant, was oral administrated to S180 tumor bearing mice in order to eliminate redundant ROS in the host and evaluate the effect against thymic apoptosis. Intact thymic structure and less apoptosis of thymocytes demonstrated that partial restoration of thymuses in S180 tumor bearing mice after 3­week VE treatment. Besides, VE treatment normalized ROS level, MDA level and SOD level of S180 tumor bearers. Thus, it could be concluded that thymic involution prevented by VE treatment in S180 tumor bearing mice was mainly due to inhibition of apoptosis in thymus and elimination of ROS over­production in the host.

In vivo induction of apoptosis by fucoxanthin, a marine carotenoid, associated with down-regulating STAT3/EGFR signaling in sarcoma 180 (S180) xenografts-bearing mice.

Previous in vitro researches have showed that fucoxanthin, a natural carotenoid isolated from sargassum, can inhibit proliferation or induce apoptosis in human neuroblastoma, hepatoma, leukemia, colon carcinoma, prostate cancer or urinary bladder cancer cells. But the precise mechanism by which fucoxanthin exerts anticarcinogenic effects is not yet fully understood. In this study, we performed an in vivo study to investigate the anti-tumor effect and mechanisms of fucoxanthin on xenografted sarcoma 180 (S180) in mice. Results revealed that fucoxanthin significantly inhibited the growth of sarcoma at the dose of 50 or 100 mg/kg. TUNEL analysis showed that the number of positive cells in the fucoxanthin-treated group was higher than that in the control group. Western blotting analysis also revealed the suppressed expression of bcl-2 and enhanced expression of cleaved caspase-3 by fucoxanthin. In addition, immunohistochemistry analysis and Western blotting analysis showed that fucoxanthin significantly decreased the expressions of survivin and vascular endothelial growth factor (VEGF). Most importantly, fucoxanthin inhibited the expressions of the epidermal growth factor receptor (EGFR) and STAT3 and phosphorylated STAT3 proteins. These results indicated that in vivo induction of apoptosis by fucoxanthin is associated with down-regulating STAT3/EGFR signaling in S180 xenografts-bearing mice.

Anti-tumor activity and immunogenicity of a succinoglycan riclin.

Natural polysaccharides have attracted considerable interests due to diverse biological activities. Succinoglycan is an extracellular polysaccharide produced by most Agrobacterium strains. Here, we confirmed riclin was a typical succinoglycan by NMR and methylation analysis, and investigated the antitumor effects of riclin in sarcoma 180 tumor-bearing mice. The results showed that riclin inhibited the tumor growth significantly as well as cyclophosphamide (CTX). While CTX caused serious damage to spleen structure, riclin increased the spleen index and promoted lymphocytes proliferation in peripheral blood, spleen and lymph nodes. Riclin decreased splenocytes apoptosis as evidenced by alterations of B-cell lymphoma-2 family proteins and Cleaved Caspase-3 protein. Moreover, 1H nuclear magnetic resonance (NMR)-based metabolomics analysis revealed that riclin partially altered the metabolic profiles of splenocytes. In conclusion, riclin is a succinoglycan that performed strong immunogenicity and suppressed sarcoma growth in mice. Succinoglycan riclin could be a potential antitumor agent for functional food and pharmaceutical purpose.

The spectrum of resistance in SR/CR mice: the critical role of chemoattraction in the cancer/leukocyte interaction.

BACKGROUND: Spontaneous regression/complete resistance (SR/CR) mice are a unique colony of mice that possess an inheritable, natural cancer resistance mediated primarily by innate cellular immunity. This resistance is effective against sarcoma 180 (S180) at exceptionally high doses and these mice remain healthy. METHODS: In this study, we challenged SR/CR mice with additional lethal transplantable mouse cancer cell lines to determine their resistance spectrum. The ability of these transplantable cancer cell lines to induce leukocyte infiltration was quantified and the percentage of different populations of responding immune cells was determined using flow cytometry. RESULTS: In comparison to wild type (WT) mice, SR/CR mice showed significantly higher resistance to all cancer cell lines tested. However, SR/CR mice were more sensitive to MethA sarcoma (MethA), B16 melanoma (B16), LL/2 lung carcinoma (LL/2) and J774 lymphoma (J774) than to sarcoma 180 (S180) and EL-4 lymphoma (EL-4). Further mechanistic studies revealed that this lower resistance to MethA and LL/2 was due to the inability of these cancer cells to attract SR/CR leukocytes, leading to tumor cell escape from resistance mechanism. This escape mechanism was overcome by co-injection with S180, which could attract SR/CR leukocytes allowing the mice to resist higher doses of MethA and LL/2. S180-induced cell-free ascites fluid (CFAF) co-injection recapitulated the results obtained with live S180 cells, suggesting that this chemoattraction by cancer cells is mediated by diffusible molecules. We also tested for the first time whether SR/CR mice were able to resist additional cancer cell lines prior to S180 exposure. We found that SR/CR mice had an innate resistance against EL-4 and J774. CONCLUSIONS: Our results suggest that the cancer resistance in SR/CR mice is based on at least two separate processes: leukocyte migration/infiltration to the site of cancer cells and recognition of common surface properties on cancer cells. The infiltration of SR/CR leukocytes was based on both the innate ability of leukocytes to respond to chemotactic signals produced by cancer cells and on whether cancer cells produced these chemotactic signals. We found that some cancer cells could escape from SR/CR resistance because they did not induce infiltration of SR/CR leukocytes. However, if infiltration of leukocytes was induced by co-injection with chemotactic factors, these same cancer cells could be effectively recognized and killed by SR/CR leukocytes.

Proteomic analysis reveals upregulation of RKIP in S-180 implanted BALB/C mouse after treatment with ascorbic acid.

Tumor cells have an invasive and metastatic phenotype that is the main cause of death for cancer patients. Tumor establishment and penetration consists of a series of complex processes involving multiple changes in gene expression. In this study, intraperitoneal administration of a high concentration of ascorbic acid inhibited tumor establishment and increased survival of BALB/C mice implanted with S-180 sarcoma cancer cells. To identify proteins involved in the ascorbic acid-mediated inhibition of tumor progression, changes in the liver proteome associated with ascorbic acid treatment of BALB/C mice implanted with S-180 were investigated using two-dimensional gel electrophoresis and mass spectrometry. Eleven protein spots were identified whose expression was different between control and ascorbic acid treatment groups. In particular, Raf kinase inhibitory protein (RKIP) and annexin A5 expression were quantitatively up-regulated. The increase in RKIP protein level was detected in the tumor tissue and accompanied by an increase in mRNA level. Our results suggest a possibility that these proteins are related to the ascorbic acid-mediated suppression of tumor formation.

Basic-helix-loop-helix (bHLH) transcription factor DEC2 negatively regulates vascular endothelial growth factor expression.

DEC1 (BHLHB2/Sharp2/Stra13) and DEC2 (BHLHB3/Sharp1) are basic-helix-loop-helix (bHLH) transcription factors, involved in cellular differentiation, responses to hypoxia and circadian rhythms. We recently showed that the expression of DEC1 and DEC2 was up-regulated by hypoxia; however, the functions of these two factors under hypoxic conditions have not been elucidated in detail. It is well established that the expression of vascular endothelial growth factor (VEGF) is up-regulated by hypoxia, and the expression of VEGF in response to hypoxia depends on transcriptional activation by a heterodimer comprising hypoxia-inducible factor 1alpha (HIF-1alpha) and arylhydrocarbon receptor nuclear translocator 1 (ARNT1). In the present study, we showed that DEC2, but not DEC1, suppressed VEGF gene expression under hypoxic conditions. DEC2 protein was co-immunoprecipitated with HIF-1alpha but not with ARNT1. The binding of HIF-1alpha to the hypoxia response element (HRE) in the VEGF promoter was decreased by DEC2 over-expression, and increased by DEC2 knockdown. We also showed that the circadian expression of VEGF showed a reciprocal pattern to that of DEC2 in cartilage. DEC2 had a circadian oscillation in implanted Sarcoma 180 cells. We conclude that DEC2 negatively regulates VEGF expression and plays an important role in the pathological conditions in which VEGF is involved.

[Research of GFP-expressing H22 cells and S180 cells by electroblot in vitro and in vivo].

Mouse hepatoma H22 cells and sarcoma cells (H22 and S180) were infected with EGFP-N1 by electroblot, and the acquired gfp-H22 and gfp-S180 cells expressing strong green fluorescence protein (GFP) fluorescence were supplemented with medium G418 Sigma (800 mg/ml). Meanwhile, the models bearing cancer (gfp H22 and gfp S180) subcutaneously and with abdominal cavity were established. There were no statistically significant differences by comparison on the cell phenotype, ultramicrostructure, growth curve and bearing cancer time between the H22 cells and S180 cells (P>0.05). The GFP fluorescence was detected with whole body GFP imaging system in vivo and with fluorescence microscope. According to the results of in vitro and in vivo assay, it was shown that, by application of fluorescence technology, the GFP-expressing H22 cells and S180 cells could be used in further studies on the tumor biological behavior.

Analysis in vivo of antitumor activity, cytotoxicity and Interaction between plasmid DNA and the cis-dichloro-tetra-amine-ruthenium(III) chloride.

Several metallic compounds recognized as potent antitumor agents, have been developed and tested in vivo and in vitro. In this work, we evaluated the toxic, therapeutic, and cytotoxic properties of the cis-dichloro-tetra-amine-ruthenium(III) chloride. Transplanted animals with Sarcoma 180 cells were treated with ruthenium(III) complex and injected i.p., at different time intervals. After the 15th day, tumoral postimplant, the animals were sacrificed and their lungs, kidneys, liver, and tumors were removed and processed for histopathological analysis. Blood samples were also taken for haematological and biochemical analyses. Interaction between the ruthenium complex and the DNA was also investigated. Besides being cytotoxic for the S180 cells, the metallic compound induced tumoral volume reduction and increased survival time of the animals treated. Serum levels of LDH, creatinine, and bilirubin increased, but no serious irreversible histopathological alterations were observed in the analyzed tissues. The compound did not cause anemia, but reduced the number of leukocytes in the treated animals. The absence of viable S180 cells, necrotic cells, and the presence of granulation tissue were observed in tumor tissue of treated animals. The Ru(III) complex, in the presence of the reduction agent, caused plasmid DNA to fragment. These results suggest that cis-RuCl(2)(NH(3))(4)Cl compound is a potent antitumoral drug in vitro and in vivo, which seems to involve binding to DNA molecule.

Study on antitumor, antioxidant and immunoregulatory activities of the purified polyphenols from pinecone of Pinus koraiensis on tumor-bearing S180 mice in vivo.

Pinecone polyphenols are bioactive dietary constituents that enhance health and help prevent and treat cancer through improving antioxidant and immunoregulatory activities. This study was designed to investigate the antitumor, antioxidant and immunoregulatory activities of the 40% ethanol eluent of polyphenols from pinecone of pinus koraiensis (PPP-40) in Sarcoma 180 (S180)-bearing mice models in vivo. The results of antitumor activity indicated that PPP-40 significantly inhibited S180 tumor growth and the dose of 150mg/kg exhibited the highest antitumor activity. Moreover, TdT-mediated dUTP nick end labeling (TUNEL) assay results further confirmed the apoptosis of S180 tumor cells. In addition, PPP-40 could obviously promote the expressions of Bax protein and inhibit the Bcl-2 protein, accordingly improve the expressions of activated Caspase-3 as well, which resulted in the activation of mitochondrial apoptotic pathway of tumor cells in S180 mice eventually. The results of antioxidant activity showed that the S180 mice treated with PPP-40 had the higher superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) activities, the more glutathione (GSH) content, and the lower malondialdehyde (MDA) level in plasma comparing with non-treated control group. Moreover, the administration with PPP-40 (150mg/kg) significantly accelerated the proliferation of splenocytes (p<0.01) and increased the monocyte phagocytosis activity in vivo simultaneously. These results revealed that PPP-40 exerts an effective antitumor activity by activating the mitochondrial apoptotic pathway and improving the antioxidant and immunoregulatory activities.

Gene therapeutic exploration: retrovirus-mediated soluble vascular endothelial growth factor receptor-2 (sFLK-1) inhibits the tumorigenicity of S180, MCF-7, and B16 cells in vivo.

Inhibition of tumor angiogenesis is an anticancer strategy in which neovasculature is targeted because tumor progression relies on neovascularization. The soluble, truncated form of vascular endothelial growth factor receptor-2 (VEGFR-2), sFLK-1, is a well-known inhibitor of endothelial cells. This kind of soluble receptor retains its high-affinity binding to VEGF, but cannot work with the receptor tyrosine transphosphorylation and activation of downstream signal transduction to induce endothelial proliferation due to the lack of the tyrosine kinase domain. Therefore, we tried to use this sFLK-1 as an inhibitor for malignant tumor gene therapy. In this study we transferred a soluble VEGFR-2 (sFLK-1) from embryo mouse liver by RT-PCR to PA317 cells through retroviral vector (pLXSN) and obtained stable expression. NIH3T3 cells were used for measuring the virus titer. The virus titer in this experiment was 2 x 10(7) CFU/ml. After 7 days of preparing subcutaneous tumor models bearing S180, MCF-7, and B16 cells in mice, respectively, 2 x 10(7) PFU of recombinant retroviruses were injected locally into the tumors the treatment groups. After treatment, the tumor size and weight were significantly smaller than that of control (p < 0.05). After autopsy, the metastasic focus numbers in the treatment groups were also less than control groups. We also measured VEGFR-2 expression in tumor tissues by Western blot to check if sFLK-1 had been integrated into the cells of tumor tissues. Expression in the treatment groups was significantly greater than that of control groups (p < 0.001). Microvessel density (MVD) and proliferative cell nuclear antigen (PCNA) were investigated to determine whether the Re-sFLK-1 fragment had the ability to inhibit tumor angiogenesis and proliferation in mice bearing S180 and MCF-7 cells. The results showed that MVD and PCNA in th e treatment groups werelower than in control groups. There were significant difference between treatment groups and control groups (p < 0.0001). The results indicated that retroviral-mediated sFLK-1 gene therapy in animal tumor models has significant therapeutic effect.

[Study on RAPD characteristics of five S180 clonal strains and on biological characteristics of passage cells from S180-S2D9].

OBJECTIVE: To study the random amplified polymorphic DNA (RAPD) characteristics of five S180, clonal cell strains with 23 primers and the biological characteristics of passage cells from S180-S2D9. METHODS: The DNA of 5 clone strains of S180 including S180-S2D9, S180-S2D7, S180-S1F11, S180-S1H10 and S180-S1B11, was amplified with RAPD-PCR using 23 single primers. The PCR products were resolved with electrophoresis on agarose gel. To analyze genomic DNA with RAPD, the cultured cells in vitro were treated with colchicine for 6 hours. Then, the chromosome numbers of 100 cells were examined under the microscope. The KM mice were injected intraperitoneally with 0.2 ml solution of S180-S2D9 cell, and the growth of ascitic fluid and the life span of the mice were observed. The cultured cells were fixed with 750 ml/L ethanol, and DNA analysis was made by Flow Cytometry. RESULTS: Three of the 23 single primers resulted in diversity between amplified products from different clonal strains. RAPD character of S180-S2D9 was analyzed by the 3 single primers; RAPD bands of the first generation, 25th generation, 50th generation and 75th generation showed no difference; ANOVA showed that the average numbers of chromosomes of four generations were of no significant difference (P>0.05). The ascites of the KM mice subjected to the intraperitoneal injection of first generation and 50th generation S180-S2D9 cells was bloody, and the survival days of mice were 13-23 d and 13-20 d respectively; ANOVA showed there was no significant difference between the first generation and 50th generation (P>0.05). DNA contents assayed by flow cytometry revealed that DNA corresponding content of the cells is individually 0.3890, 0.3542, 0.3575 and 0. 3984. These results imply that the S180-S2D9 passage cells have not been found to vary a lot. CONCLUSION: It is adaptable to give quality control to the clonal cell strains with RAPD.

Antitumor, genotoxicity and anticlastogenic activities of polysaccharide from Curcuma zedoaria.

The antitumor effect of the partially purified polysaccharide from Curcuma zedoaria was studied in mice transplanted with sarcoma 180 cells. The polysaccharide fraction, CZ-1-III, at dose of 6.25 mg/kg/d showed 50% inhibition in solid tumor growth. When mice were injected with fractions, CZ-1 and CZ-1-III, at the dose of 100.0 mg/kg, 91.6% and 97.1% of tumor growth were inhibited, respectively, indicating that the cytotoxic effect of polysaccharide on sarcoma 180 cells increases upon increasing the amount of polysaccharide administered. To assess the genotoxicity of CZ-1-III fraction, several classical toxicological tests were performed. In Ames test, CZ-1-III did not show any transformation of revertant with or without S-9 metabolic activating system, indicating the lack of mutagenic effect of the compound. To assess clastogenic effect, micronucleus and chromosomal aberration assays were performed using Chinese hamster lung (CHL) fibroblast cells. However, up to 259.0 microg/ml concentration of CZ-1-III, neither micronucleus formation nor chromosomal aberration was induced regardless of the presence of S-9 metabolic activating system. Inhibition of CZ-1-III on micronucleus formation induced by mitomycin C was exhibited in a dose-dependent manner, maximally up to 52.0%. These results strongly suggest that CZ-1-III, the polysaccharide fraction from C. Zedoaria, decreases tumor size of mouse and prevents chromosomal mutation.

Induced multidrug-resistance in murine sarcoma 180 cells grown in vitro and in vivo and associated changes in expression of multidrug-resistance DNA-sequences and membrane glycoproteins.

The aim of this investigation was to analyze the resistance to doxorubicin and daunorubicin of murine sarcoma 180 cells grown in vitro (monolayer) and in vivo (ascites form). The colchicine-resistant CHO cells were used as controls. A multidrug-resistant phenotype was found in all investigated cell lines. Multidrug resistant cells grown in tissue culture or as ascites tumor cells needed more time to accumulate rhodamine 123 than their sensitive parental cells. In order to evaluate whether the resistant cells show alterations in the plasma membranes, different methods were applied (immunocytochemistry, immunofluorescence, immunoblotting) using the monoclonal antibodies 265/F4 and C 219. These methods all revealed an increased expression of the glycoprotein Mr 170 kd in the multidrug-resistant cell lines. To determine whether multidrug DNA sequences were expressed in the resistant cell lines, slot blots and Northern blots with RNA of sensitive and resistant cells were performed using the clones pDR 7.8 and pcDR 1.5. Elevated RNA levels were detected in all resistant cell lines.

Rifampicin-induced chromatid exchanges in Sarcoma-180 mouse ascites cells.

Sarcoma-180 ascites tumour-bearing male mice were injected i.p. with single dose of 0.5, 2.5, 5 mg/kg body wt. of rifampicin. Cells were sampled for mitotic chromosome analysis 4, 16 or 24 h after treatment. The maximal yield of chromatid-type aberrations induced was found 24 h after treatment with 5 mg/kg of rifampicin. More than 60% of the cells carried at least one chromatid exchange. The majority of these were exchanges derived from breaks in the centromeric heterochromatin.

Involvement of NOR-bearing chromosome in Rb-fusion: evidence from an in vivo murine sarcoma cell line.

The involvement of NOR-bearing chromosome in the formation of two stable and transmissible Robertsonian markers is reported in ascitic form of mouse Sarcoma 180 (S180) adapted in vivo to outbred strain of Swiss albino mice. The chromosomes involved in Robertsonian fusion included t(8;12) and t(16;17).

Changes in the template activity of chromatin isolated from sarcoma-180 ascites cells treated with mitomycin C and gamma irradiation in vivo.

The murine ascites sarcoma 180 cells were used to test the in vivo effectiveness of mitomycin C (MMC) and gamma-radiation applied in combination. The action of intraperitoneal administration of MMC and/or whole-body gamma irradiation on sarcoma 180 tumor bearing Swiss albino mice was investigated by studying the template activity of isolated tumor chromatin. The Km value for transcription of 10 Gy-irradiated chromatin was found to decrease with time implying an increase in the template efficiency with respect to that of the unirradiated control. Maximum decrease in Km was observed after 24 h of irradiation. MMC treatment (7 mg/kg body weight of mouse) for 18 h resulted in an inhibition of the transcription rate. Severe inhibition in the template activity was found when cells were subjected to MMC treatment 18 h prior to irradiation with 10 Gy. Susceptibility of tumor chromatin to DNase II followed the same pattern as observed in the case of transcription indicating structural alteration of the treated chromatin. The data showed that DNA damage and its consequences produced in the ascites cells by prior treatment of MMC were not repaired during the 18 h period after which the application of radiation enhanced cytotoxicity.

[Molecular biological evidence for the genetic stability of in vivo passaged doxorubicin resistant cell line S-180R].

OBJECTIVE: To assess the genetic stability of doxorubicin resistant sarcoma 180 cell line(S-180R) after in vivo passages. METHODS: Flow cytometry, Southern blot, Northern blot and RT-PCR were used to examine genes and molecules related to drug resistance. RESULTS: The drug-efflux of S-180R was nearly 100-fold as high as that of the parental cells. The ratio of half peak width to peak height was 0.23 as compared to 0.56 measured two years before when the S-180R cell line was initially established. The mdr-1 gene was significantly amplified and transcribed while the transcription of topoisomerase II alpha gene was decreased. However there was no increase in mRNA expression of the multidrug resistance associated protein(MRP). CONCLUSION: Compared with the initially established S-180R, its resistance to doxorubicin is not only maintained but in fact has been increased since in vivo passage for 2 years. The major mechanism is amplification and over-expression of mdr-1 gene, but decreased topoisomerase II alpha also contributes. S-180R is an ideal experimental model for the study of doxorubicin resistance and its reversion.