Significant blockade of multiple receptor tyrosine kinases by MGCD516 (Sitravatinib), a novel small molecule inhibitor, shows potent anti-tumor activity in preclinical models of sarcoma.

Sarcomas are rare but highly aggressive mesenchymal tumors with a median survival of 10-18 months for metastatic disease. Mutation and/or overexpression of many receptor tyrosine kinases (RTKs) including c-Met, PDGFR, c-Kit and IGF1-R drive defective signaling pathways in sarcomas. MGCD516 (Sitravatinib) is a novel small molecule inhibitor targeting multiple RTKs involved in driving sarcoma cell growth. In the present study, we evaluated the efficacy of MGCD516 both in vitro and in mouse xenograft models in vivo. MGCD516 treatment resulted in significant blockade of phosphorylation of potential driver RTKs and induced potent anti-proliferative effects in vitro. Furthermore, MGCD516 treatment of tumor xenografts in vivo resulted in significant suppression of tumor growth. Efficacy of MGCD516 was superior to imatinib and crizotinib, two other well-studied multi-kinase inhibitors with overlapping target specificities, both in vitro and in vivo. This is the first report describing MGCD516 as a potent multi-kinase inhibitor in different models of sarcoma, superior to imatinib and crizotinib. Results from this study showing blockade of multiple driver signaling pathways provides a rationale for further clinical development of MGCD516 for the treatment of patients with soft-tissue sarcoma.

Nitroxoline impairs tumor progression in vitro and in vivo by regulating cathepsin B activity.

Cathepsin B is a ubiquitously expressed lysosomal cysteine protease that participates in protein turnover within lysosomes. However, its protein and activity levels have been shown to be increased in cancer. Cathepsin B endopeptidase activity is involved in the degradation of extracellular matrix, a process that promotes tumor invasion, metastasis and angiogenesis. Previously, we reported an established antibiotic nitroxoline as a potent and selective inhibitor of cathepsin B. In the present study, we elucidated its anti-tumor properties in in vitro and in vivo tumor models. Tumor and endothelial cell lines with high levels of active cathepsin B were selected for functional analysis of nitroxoline in vitro. Nitroxoline significantly reduced extracellular DQ-collagen IV degradation by all evaluated cancer cell lines using spectrofluorimetry. Nitroxoline also markedly decreased tumor cell invasion monitored in real time and reduced the invasive growth of multicellular tumor spheroids, used as a 3D in vitro model of tumor invasion. Additionally, endothelial tube formation was significantly reduced by nitroxoline in an in vitro angiogenesis assay. Finally, nitroxoline significantly abrogated tumor growth, angiogenesis and metastasis in vivo in LPB fibrosarcoma and MMTV-PyMT breast cancer mouse models. Overall, our results designate nitroxoline as a promising drug candidate for anti-cancer treatment.

The essential role of PIM kinases in sarcoma growth and bone invasion.

PIM kinases are a family of serine/threonine kinases composed of three different isoforms (PIM1, PIM 2 and PIM 3) that are highly homologous. Their expression is mediated by the JAK/STAT signalling pathway, providing survival and cell cycle transition signals. PIM kinases are heavily targeted for anticancer drug discovery. However, very little is known about the relative contribution of the different isoforms to the tumourigenesis process in vivo, and how their individual inhibition might affect tumour growth. Taking advantage of genetically modified mice, we explored whether the inhibition of specific isoforms is required to prevent sarcomas induced by 3-methylcholanthrene carcinogenic treatment. We found that absence of Pim2 and Pim3 greatly reduced sarcoma growth to a similar extent to the absence of all three isoforms. This model of sarcoma generally produces bone invasion by the tumour cells. Lack of Pim2 and Pim3 reduced tumour-induced bone invasion by 70%, which is comparable with the reduction of tumour-induced bone invasion in the absence of all three isoforms. Similar results were obtained in mouse embryonic fibroblasts (MEFs) derived from these knockout (KO) mice, where double Pim2/3 KO MEFs already showed reduced proliferation and were resistant to oncogenic transformation by the RAS oncogene. Our data also suggest an important role of Gsk3ß phosphorylation in the process of tumourigenesis.

Tempol reduces the therapeutic effect of cyclophosphamide on an experimental tumour model.

The nitroxyl radical 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol) is reported to elicite some effects on different biological models. This paper studied the influence of Tempol on the therapeutic action of an alkylating agent cyclophosphamide (CP) in the transplantable murine lymphosarcoma LS. When administered exactly before CP, Tempol exerted no influence on efficacy of the tumour therapy but significantly reduced it under a single or multiple preliminary injections 1-3 days before CP. This regimen of Tempol administration is found to elevate the activity of aldehyde dehydrogenase in the liver, the enzyme which is known to reduce the yield of the activated metabolite(s) of CP.

The transcriptional cascade associated with creatine kinase down-regulation and mitochondrial biogenesis in mice sarcoma.

The tissue-specific expressions of creatine kinase (CK) isoforms are regulated by the coordinated action of various transcription factors. The myogenic differentiation factor D (MyoD) family of proteins and the myocyte-specific enhancer binding factor 2 family of transcription factors are important in regulating the muscle-specific expression of cytosolic muscle-type CK (MCK) and mitochondrial CKs. As reported in some related studies, TNF-alpha mediated degradation of MyoD and myogenin mRNA may lead to severe muscle wasting and cachexia, which is characterized by a low transcript level of MCK and myosin heavy chain proteins. In our previous study, we reported on a complete loss of total CK activity and expression when sarcoma was induced in mouse skeletal muscle (Patra et al. FEBS J. 275 (2008) 3236-3247). This study aimed at investigating the transcriptional cascade of CK down-regulation in carcinogen-induced sarcoma in mouse muscle. Both CK deficiency and enhanced nitric oxide synthase (NOS) were known to augment mitochondrial biogenesis, so we also explored the activation of the transcriptional cascade of mitochondrial biogenesis in this cancer. We observed the activation of the TNF-alpha-mediated nitric oxide production pathway with NFkappaB activation and concomitant degradation of MyoD and myogenin mRNA. Exploration of mitochondrial biogenesis revealed high cytochrome c oxidase activity and mitochondrial DNA content in sarcoma. The PGC-related co-activator seems to have a major role in regulating mitochondrial biogenesis by upregulating nuclear respiratory factors and mitochondrial transcription factor A. From the above findings, it can be concluded that severe muscle degeneration leads to CK down-regulation in sarcoma, and that the stimulation of mitochondrial biogenesis indicated a scenario representing both CK deficiency and NOS overexpression on the one hand, and altered bioenergetic profiling on the other.

Amifostine induces antioxidant enzymatic activities in normal tissues and a transplantable tumor that can affect radiation response.

PURPOSE: To determine whether amifostine can induce elevated manganese superoxide dismutase (SOD2) in murine tissues and a transplantable SA-NH tumor, resulting in a delayed tumor cell radioprotective effect. METHODS AND MATERIALS: SA-NH tumor-bearing C3H mice were treated with a single 400 mg/kg or three daily 50 mg/kg doses of amifostine administered intraperitoneally. At selected time intervals after the last injection, the heart, liver, lung, pancreas, small intestine, spleen, and SA-NH tumor were removed and analyzed for SOD2, catalase, and glutathione peroxidase (GPx) enzymatic activity. The effect of elevated SOD2 enzymatic activity on the radiation response of SA-NH cells was determined. RESULTS: SOD2 activity was significantly elevated in selected tissues and a tumor 24 h after amifostine treatment. Catalase and GPx activities remained unchanged except for significant elevations in the spleen. GPx was also elevated in the pancreas. SA-NH tumor cells exhibited a twofold elevation in SOD2 activity and a 27% elevation in radiation resistance. Amifostine administered in three daily fractions of 50 mg/kg each also resulted in significant elevations of these antioxidant enzymes. CONCLUSIONS: Amifostine can induce a delayed radioprotective effect that correlates with elevated levels of SOD2 activity in SA-NH tumor. If limited to normal tissues, this delayed radioprotective effect offers an additional potential for overall radiation protection. However, amifostine-induced elevation of SOD2 activity in tumors could have an unanticipated deleterious effect on tumor responses to fractionated radiation therapy, given that the radioprotector is administered daily just before each 2-Gy fractionated dose.

Progressive decrease of phosphocreatine, creatine and creatine kinase in skeletal muscle upon transformation to sarcoma.

In vertebrates, phosphocreatine and ATP are continuously interconverted by the reversible reaction of creatine kinase in accordance with cellular energy needs. Sarcoma tissue and its normal counterpart, creatine-rich skeletal muscle, are good source materials to study the status of creatine and creatine kinase with the progression of malignancy. We experimentally induced sarcoma in mouse leg muscle by injecting either 3-methylcholanthrene or live sarcoma 180 cells into one hind leg. Creatine, phosphocreatine and creatine kinase isoform levels decreased as malignancy progressed and reached very low levels in the final stage of sarcoma development; all these parameters remained unaltered in the unaffected contralateral leg muscle of the same animal. Creatine and creatine kinase levels were also reduced significantly in frank malignant portions of human sarcoma and gastric and colonic adenocarcinoma compared with the distal nonmalignant portions of the same samples. In mice, immunoblotting with antibodies against cytosolic muscle-type creatine kinase and sarcomeric mitochondrial creatine kinase showed that both of these isoforms decreased as malignancy progressed. Expressions of mRNA of muscle-type creatine kinase and sarcomeric mitochondrial creatine kinase were also severely downregulated. In human sarcoma these two isoforms were undetectable also. In human gastric and colonic adenocarcinoma, brain-type creatine kinase was found to be downregulated, whereas ubiquitous mitochondrial creatine kinase was upregulated. These significantly decreased levels of creatine and creatine kinase isoforms in sarcoma suggest that: (a) the genuine muscle phenotype is lost during sarcoma progression, and (b) these parameters may be used as diagnostic marker and prognostic indicator of malignancy in this tissue.

R16, a novel amonafide analogue, induces apoptosis and G2-M arrest via poisoning topoisomerase II.

Amonafide, a naphthalimide derivative, although selected for exploratory clinical trials for its potent anticancer activity, has long been challenged by its unpredictable side effects. In the present study, a novel amonafide analogue, 2-(2-dimethylamino)-6-thia-2-aza-benzo-[def]-chrysene-1,3-diones (R16) was synthesized by substituting 5'-NH(2) of the naphthyl with a heterocyclic group to amonafide, with additional introduction of a thiol group. In a panel of various human tumor cell lines, R16 was more cytotoxic than its parent compound amonafide. It was also effective against multidrug-resistant cells. Importantly, the i.p. administration of R16 inhibited tumor growth in mice implanted with S-180 sarcoma and H(22) hepatoma. The molecular and cellular machinery studies showed that the R16 functions as a topoisomerase II (topo II) poison via binding to the ATPase domain of human topo IIalpha. The superior cytotoxicity of R16 to amonafide was ascribed to its potent effects on trapping topo II-DNA cleavage complexes. Moreover, using a topo II catalytic inhibitor aclarubicin, ataxia-telangiectasia-mutated (ATM)/ATM- and Rad3-related (ATR) kinase inhibitor caffeine and topo II-deficient HL-60/MX2 cells, we further showed that R16-triggered DNA double-strand breaks, tumor cell cycle arrest, and apoptosis were in a topo II-dependent manner. Taken together, R16 stood out by its improved anticancer activity, appreciable anti-multidrug resistance activities, and well-defined topo II poisoning mechanisms, as comparable with the parent compound amonafide. All these collectively promise the potential value of R16 as an anticancer drug candidate, which deserves further development.

Enzymatic processing of collagen IV by MMP-2 (gelatinase A) affects neutrophil migration and it is modulated by extracatalytic domains.

Proteolytic degradation of basement membrane influences the cell behavior during important processes, such as inflammations, tumorigenesis, angiogenesis, and allergic diseases. In this study, we have investigated the action of gelatinase A (MMP-2) on collagen IV, the major constituent of the basement membrane. We have compared quantitatively its action on the soluble forms of collagen IV extracted with or without pepsin (from human placenta and from Engelbreth-Holm-Swarm [EHS] murine sarcoma, respectively). The catalytic efficiency of MMP-2 is dramatically reduced in the case of the EHS murine sarcoma with respect to the human placenta, probably due to the much tighter packing of the network which renders very slow the speed of the rate-limiting step. We have also enquired on the role of MMP-2 domains in processing collagen IV. Addition of the isolated collagen binding domain, corresponding to the fibronectin-like domain of whole MMP-2, greatly in hibits the cleavage process, demonstrating that MMP-2 interacts with collagen type IV preferentially through its fibronectin-like domain. Conversely, the removal of the hemopexin-like domain, using only the catalytic domain of MMP-2, has only a limited effect on the catalytic efficiency toward collagen IV, indicating that the missing domain does not have great relevance for the overall mechanism. Finally, we have investigated the effect of MMP-2 proteolytic activity ex vivo. MMP-2 action negatively affects the neutrophils' migration across type IV coated membranes and this is likely related to the production of lower molecular weight fragments that impair the cellular migration.

[Dynamics of free-radical oxidation processes in transplantable sarcoma-45 as indicator of tumor and body interaction].

Induced chemoluminescence was used for 30 days to measure lipids peroxidation in transplantable sarcoma-45 homogenates in 83 noninbred male rats. It was found that tumor/body interaction can be interpreted in terms of free-radical oxidation processes taking place in tumor tissue. In the middle of the experiment, the kinetics of chemoluminescence soared up which was accompanied by slight increase in tumor mass. Later on, tumor growth involved a rise in antioxidant enzyme levels which is supposedly accounted for by the expression of the genes responsible for the rate of antioxidant reactions in general. That increase resulted in uncontrollable growth of tumor.

Cyclophilin A-deficient mice are resistant to immunosuppression by cyclosporine.

Cyclosporine is an immunosuppressive drug that is widely used to prevent organ transplant rejection. Known intracellular ligands for cyclosporine include the cyclophilins, a large family of phylogenetically conserved proteins that potentially regulate protein folding in cells. Immunosuppression by cyclosporine is thought to result from the formation of a drug-cyclophilin complex that binds to and inhibits calcineurin, a serine/threonine phosphatase that is activated by TCR engagement. Amino acids within the cyclophilins that are critical for binding to cyclosporine have been identified. Most of these residues are highly conserved within the 15 mammalian cyclophilins, suggesting that many are potential targets for the drug. We examined the effects of cyclosporine on immune cells and mice lacking Ppia, the gene encoding the prototypical cyclophilin protein cyclophilin A. TCR-induced proliferation and signal transduction by Ppia(-/-) CD4(+) T cells were resistant to cyclosporine, an effect that was attributable to diminished calcineurin inhibition. Immunosuppressive doses of cyclosporine failed to block the responses of Ppia(-/-) mice to allogeneic challenge. Rag2(-/-) mice reconstituted with Ppia(-/-) splenocytes were also cyclosporine resistant, indicating that this property is intrinsic to Ppia(-/-) immune cells. Thus, among multiple potential ligands, CypA is the primary mediator of immunosuppression by cyclosporine.

NOS isoenzyme content in brain nuclei as related to food intake in experimental cancer cachexia.

Evidence implies that nitric oxide (NO) in the central nervous systems mediates anorexia in tumor-bearing hosts. We have therefore evaluated, by immunohistochemical image analyses, net alterations of nitric oxide synthases (nNOS, eNOS, iNOS) in brain nuclei [paraventricular hypothalamic nucleus (PVN), medial habenular nucleus (MHB), lateral habenular nucleus (LHB), paraventricular thalamic nucleus (PV), lateral hypothalamic area (LHA), ventromedial hypothalamic nucleus (VMH), nucleus of the solitary tract (NTS)] of tumor-bearing mice (TB) with prostanoid-related anorexia. Pair-fed (PF) and freely fed (FF) non-tumor-bearing mice were used as controls. c-fos was analyzed as indicator of neuronal activation. nNOS was significantly increased in VMH and PVN from TB mice, while eNOS was significantly increased in LHB and LHA. iNOS was significantly increased in LHA and PVN nuclei, but decreased in MHB, LHB and VMH from tumor-bearers. However, several of these alterations were similarly observed in brain nuclei from pair-fed controls. Provision of unspecific NOS-antagonists to TB mice increased nNOS, eNOS and iNOS in several brain nuclei (PVN, LHA, VMH), but left tumor-induced anorexia unchanged. c-fos was significantly increased in all brain nuclei in PF mice except for NTS, LHA and PVN compared to controls, while tumor-bearing mice had increased c-fos in LHA and PVN only compared to controls. Our results demonstrate a complex picture of NOS expression in brain areas of relevance for appetite in tumor-bearing hosts, where most changes seemed to be secondary to stress during negative energy balance. By contrast, NOS content in PVN and LHA nuclei remains candidate behind anorexia in tumor disease. However, nitric oxide does not seem to be a primary mediator behind tumor-induced anorexia. NO may rather secondarily support energy intake in conditions with negative energy balance.

Nitric oxide, a mediator of inflammation, suppresses tumorigenesis.

Inflammation influences the development of cancer. The nitric oxide synthase (NOS2) is induced by inflammatory cytokines, e.g., tumor necrosis factor alpha and interleukin 1beta, and produces nitric oxide (NO*), a critical mediator of the inflammatory response. Because p53 governs NO* production by transcriptionally transrepressing NOS2, we used a genetic strategy to determine whether NO* and p53 cooperatively regulate tumorigenesis. Lymphomas developed more rapidly in p53-/-NOS2-/- or p53-/-NOS2+/- mice than in p53-/-NOS2+/+ mice that were cross-bred into a >95% C57BL6 background and maintained in a pathogen-free condition. Likewise, sarcomas and lymphomas developed faster in p53+/-NOS2-/- or p53+/-NOS2+/- than in p53+/-NOS2+/+ mice. When compared with the double knockout mice, p53-/-NOS2+/+ mice showed a higher apoptotic index and a decreased proliferation index with an increased expression of death receptor ligands, CD95-L and tumor necrosis factor-related apoptosis-inducing ligand, and the cell cycle checkpoint protein, p21(waf1), in the spleen and thymus before tumor development. Furthermore, mice deficient in both p53 and NOS2 produced a high level of anti-inflammatory interleukin 10 when compared with p53-deficient mice. These studies provide genetic and mechanistic evidence that NO* can suppress tumorigenesis.

(-)-Epigallocatechin gallate inhibits membrane-type 1 matrix metalloproteinase, MT1-MMP, and tumor angiogenesis.

Membrane-type 1 matrix metalloproteinase (MT1-MMP), which hydrolyzes type I collagen and activates MMP-2, are deeply involved in angiogenesis as well as in tumor cell invasion and metastasis. We previously screened a number of natural and synthetic compounds to obtain a specific inhibitor of MT1-MMP and observed that (-)-epigallocatechin gallate (EGCG) has a potent and distinct inhibitory activity against MT1-MMP. In the present study, we investigated the effect of EGCG on tumor angiogenesis. EGCG significantly inhibited the invasion of human umbilical vein endothelial cells (HUVECs) at the concentration of 10 microM. This effect was not due to the toxicity of EGCG since this concentration of EGCG did not affect the HUVEC growth. Furthermore, morphological change of HUVEC at this concentration of EGCG was not observed under confocal laser scanning microscopy. EGCG suppressed tube formation by HUVECs in vitro and angiogenesis in vivo by using dorsal air sac model. Finally, we observed that both colon 26 NL17 carcinoma and Meth A sarcoma growth was suppressed in these tumor-bearing mice by EGCG administration, at least partly though the inhibition of angiogenesis.

Antigen-specific inhibition of CD8+ T cell response by immature myeloid cells in cancer is mediated by reactive oxygen species.

Tumor growth is associated with the accumulation of immature myeloid cells (ImC), which in mice are characterized by the expression of Gr-1 and CD11b markers. These cells suppress Ag-specific CD8+ T cells via direct cell-cell contact. However, the mechanism of immunosuppressive activity of tumor-derived ImC remains unclear. In this study we analyzed the function of ImC isolated from tumor-free control and tumor-bearing mice. Only ImC isolated from tumor-bearing mice, not those from their control counterparts, were able to inhibit the Ag-specific response of CD8+ T cells. ImC obtained from tumor-bearing mice had significantly higher levels of reactive oxygen species (ROS) than ImC isolated from tumor-free animals. Accumulation of H2O2, but not superoxide or NO, was a major contributor to this increased pool of ROS. It appears that arginase activity played an important role in H2O2 accumulation in these cells. Inhibition of ROS in ImC completely abrogated the inhibitory effect of these cells on T cells, indicating that ImC generated in tumor-bearing hosts suppress the CD8+ T cell response via production of ROS. Interaction of ImC with Ag-specific T cells in the presence of specific Ags resulted in a significant increase in ROS production compared with control Ags. That increase was independent of IFN-gamma production by T cells, but was mediated by integrins CD11b, CD18, and CD29. Blocking of these integrins with specific Abs abrogated ROS production and ImC-mediated suppression of CD8+ T cell responses. This study demonstrates a new mechanism of Ag-specific T cell inhibition mediated by ROS produced by ImCs in cancer.

Delayed cytoprotection after enhancement of Sod2 (MnSOD) gene expression in SA-NH mouse sarcoma cells exposed to WR-1065, the active metabolite of amifostine.

SA-NH mouse sarcoma cells were grown to confluence and then exposed to either 40 microM or 4 mM of WR-1065, i.e. the active thiol form of amifostine, for 30 min and then washed. Total RNA and protein were isolated at various times up to 24 h after exposure. Both concentrations of WR-1065 were equally effective in affecting Sod2 (also known as MnSOD) gene expression and protein levels. Northern blot analysis using a mouse cDNA probe revealed three Sod2 transcripts of 1, 4 and 6 kb. Expression of both the 4- and 6-kb transcripts increased by 20 and 60%, respectively, and remained elevated over a period of 4 to 20 h. Sod2 protein levels, as determined by Western blot analysis, increased 15-fold over background control levels over the same interval. Sod2 protein was evaluated using activity gels and was found to be active. SA-NH cells were irradiated with X rays either in the presence of 40 microM or 4 mM WR-1065 or 24 h later after its removal, when Sod2 protein levels were most elevated. No protection was observed for cells irradiated in the presence of 40 microM WR-1065. In contrast, survival after a dose of 2 Gy was elevated 1.27-, 1.14- and 1.20-fold in SA-NH cells irradiated in the presence of 4 mM WR-1065 or 24 h after exposure of the cells to 40 microM and 4 mM WR-1065, respectively. The increased survival levels observed 24 h after exposure to WR-1065 represents a delayed radioprotective effect of WR-1065 and corresponds to the time at which Sod2 protein levels are most elevated. These data demonstrate a novel mechanism for radioprotection by WR-1065 and suggest a new potential concern regarding the issue of tumor protection.

Inhibition of spontaneous metastases formation by amifostine.

Amifostine was investigated for its ability to inhibit spontaneous metastases formation using the well-characterized murine sarcoma, Sa-NH. Amifostine was administered intraperitoneally at a dose of 50 mg/kg every other day for 6 days to C3Hf/Kam mice until tumors reached an average size of 8-8.5 mm in diameter. Amifostine was again administered immediately after surgical removal of the tumor-bearing limbs by amputation, and then once more 2 days later. Twenty-one days later, animals were evaluated for the presence of spontaneously developed pulmonary metastases. Nontumor-bearing control animals were sham treated using the same dosing and surgery schedules. Treatment with amifostine appeared to slightly delay tumor growth, that is, 13 vs. 12 days for tumors to reach an average diameter of 8 mm. Amifostine reduced both the incidence of pulmonary metastases formed in experimental animals from 77% to 57% (p < 0.05), and their average number per animal from 12.8 +/- 5.4 (SEM) to 2.9 +/- 1.1 (SEM). The effect of amifostine exposure on serum levels of the angiogenesis inhibitor angiostatin was also determined using Western blot analysis. Consistent with the antimetastatic effect, exposure of animals to 50 mg/kg of amifostine resulted in a 4-fold enhanced serum level of angiostatin above control levels. This phenomenon occurred in tumor-bearing and nontumor-bearing animals. The effects of amifostine on matrix metalloproteinase (MMP) enzymatic activity was also determined using gelatin zymography. Conditioned growth medium collected from Sa-NH cells grown to confluency was exposed to various concentrations of SH, i.e., 2-[(aminopropyl)amino]ethane-thiol (WR-1065), the active thiol form of amifostine, for either 30 min or 18 hr. WR-1065, as a function of increasing dose and time, inhibited the enzymatic activities of MMP-2 and MMP-9. At a concentration and time of exposure likely to be achieved in vivo, that is, 40 microM and 30 min, MMP-2 and MMP-9 activities were reduced to between 30% and 40% of control values. Consistent with these affects, WR-1065 was also found to be effective in inhibiting the ability of Sa-NH cells to migrate through Matrigel membranes. After an 18-hr exposure under in vitro conditions, WR-1065 at concentrations of 4, 40 and 400 microM, and 4 mM, inhibited Sa-NH migration to 11%, 44%, 81% and 97% of control values, respectively. The abilities of amifostine and its active thiol WR-1065 to stimulate angiostatin production in mice, and to inhibit the MMP enzymatic activities and invasion ability of Sa-NH cells under in vitro conditions, are consistent with the observed antimetastatic effects exhibited against Sa-NH tumors growing in vivo.

Improved measurement of tyrosine-phosphorylated DNA-PKcs by isoelectric focusing as an indirect determination of DNA-PK activity in vivo.

UNLABELLED: Isolation of various phosphorylated forms of DNA-PKcs by SDS gel electrophoresis is difficult because of the same molecular weight. By isoelectric focusing (IEF), we confirmed a de-phosphorylated (pI 9.0) and a phosphorylated (pI 6.9) form of DNA-PKcs. When cells were damaged by X-irradiation (5 Gy), an additional tyrosine-phosphorylated (p-Tyr) DNA-PKcs form (pI 6.3) appeared in both nuclear and cytoplasmic fractions, after 30 minsutes. CONCLUSION: IEF in combination with Western blotting of p-Tyr-DNA-PKcs results in an appropriate separation of the p-Tyr form from other phosphorylated forms of DNA-PKcs, which may permit an indirect measurement of changes in DNA-PK activity in irradiated cells.

Neoplastic transformation and tumorigenesis associated with overexpression of phospholipase D isozymes in cultured murine fibroblasts.

Phospholipase D (PLD) has been suggested to play an important role in a variety of cellular functions. PLD activity has been shown to be significantly elevated in many tumours and transformed cells, suggesting the possibility that PLD might be involved in tumorigenesis. In this study, we have established stable cell lines overexpressing PLD1 and PLD2 from fibroblast cells. These cells, but not control cells, showed altered growth properties and anchorage-independent growth in soft agar. Both PLD1 and PLD2 also induced an up-regulation of the activity of matrix metalloprotease-9 as detected by zymograms. Furthermore, both PLD1 and PLD2 transformants, but not vector-transfectants, induced undifferentiated sarcoma when transplanted into nude mice. Both PLD1- and PLD2-mediated cell cycle distributions in stable cell lines revealed an increased fraction of cells in the S phase compared with control cells. Interestingly, the level of cyclin D3 protein, known as an activator of G(1) to S phase transition in the cell cycle, was aberrantly high in cells overexpressing PLD1 and PLD2 compared with control cells. These results suggest that overexpression of PLD isozymes may play an important role in neoplastic transformation.

Telomerase activity is not altered by regular strenuous exercise in skeletal muscle or by sarcoma in liver of rats.

Telomerase is a specialized ribonucleoprotein enzyme complex which prevents the loss of the telomere. The activity of telomerase can be up- and down-regulated by various oxidative stresses but the effect of physical exercise is not known, whereas the modifying effect of cancer on telomerase activity is well documented. In the first study, we investigated the effect of mild and strenuous exercise training on telomerase activity, assessed by a PCR ELISA kit. No alteration in telomerase activity was detected. In the second investigation, solid sarcoma cells were transplanted to control, exercise trained or exercise trained and still exercising mice. On the 16th day after the transplantation, the size of tumors in the exercise trained group was 72% and in the exercising group 57% (P < 0.05) of that in the controls. Telomerase activity and 8-hydroxy-2'-deoxyguanosine levels in the liver were not significantly altered by exercise and/or sarcoma. We conclude that mild and strenuous exercise training does not significantly affect the activity of telomerase in the systems studied. Exercise training during sarcoma significantly retards the development of tumors and could possibly serve as a positive adjunct to treatment.