Tumor Response to Radiotherapy is Dependent on Genotype-Associated Mechanisms in vitro and in vivo

Background: We have previously shown that in vitro radiosensitivity of human tumor cells segregate non-randomly into a limited number of groups. Each group associates with a specific genotype. However we have also shown that abrogation of a single gene (p21) in a human tumor cell unexpectedly sensitized xenograft tumors comprised of these cells to radiotherapy while not affecting in vitro cellular radiosensitivity. Therefore in vitro assays alone cannot predict tumor response to radiotherapy. In the current work, we measure in vitro radiosensitivity and in vivo response of their xenograft tumors in a series of human tumor lines that represent the range of radiosensitivity observed in human tumor cells. We also measure response of their xenograft tumors to different radiotherapy protocols. We reduce these data into a simple analytical structure that defines the relationship between tumor response and total dose based on two coefficients that are specific to tumor cell genotype, fraction size and total dose. Methods: We assayed in vitro survival patterns in eight tumor cell lines that vary in cellular radiosensitivity and genotype. We also measured response of their xenograft tumors to four radiotherapy protocols: 8 × 2 Gy; 2 × 5Gy, 1 × 7.5 Gy and 1 × 15 Gy. We analyze these data to derive coefficients that describe both in vitro and in vivo responses. Results: Response of xenografts comprised of human tumor cells to different radiotherapy protocols can be reduced to only two coefficients that represent 1) total cells killed as measured in vitro 2) additional response in vivo not predicted by cell killing. These coefficients segregate with specific genotypes including those most frequently observed in human tumors in the clinic. Coefficients that describe in vitro and in vivo mechanisms can predict tumor response to any radiation protocol based on tumor cell genotype, fraction-size and total dose. Conclusions: We establish an analytical structure that predicts tumor response to radiotherapy based on coefficients that represent in vitro and in vivo responses. Both coefficients are dependent on tumor cell genotype and fraction-size. We identify a novel previously unreported mechanism that sensitizes tumors in vivo; this sensitization varies with tumor cell genotype and fraction size

Changes in Mouse Thymus and Spleen after Return from the STS-135 Mission in Space

Our previous results with flight (FLT) mice showed abnormalities in thymuses and spleens that have potential to compromise immune defense mechanisms. In this study, the organs were further evaluated in C57BL/6 mice after Space Shuttle Atlantis returned from a 13-day mission. Thymuses and spleens were harvested from FLT mice and ground controls housed in similar animal enclosure modules (AEM). Organ and body mass, DNA fragmentation and expression of genes related to T cells and cancer were determined. Although significance was not obtained for thymus mass, DNA fragmentation was greater in the FLT group (P<0.01). Spleen mass alone and relative to body mass was significantly decreased in FLT mice (P<0.05). In FLT thymuses, 6/84 T cell-related genes were affected versus the AEM control group (P<0.05; up: IL10, Il18bp, Il18r1, Spp1; down: Ccl7, IL6); 15/84 cancer-related genes had altered expression (P<0.05; up: Casp8, FGFR2, Figf, Hgf, IGF1, Itga4, Ncam1, Pdgfa, Pik3r1, Serpinb2, Sykb; down: Cdc25a, E2F1, Mmp9, Myc). In the spleen, 8/84 cancer-related genes were affected in FLT mice compared to AEM controls (P<0.05; up: Cdkn2a; down: Birc5, Casp8, Ctnnb1, Map2k1, Mdm2, NFkB1, Pdgfa). Pathway analysis (apoptosis signaling and checkpoint regulation) was used to map relationships among the cancer–related genes. The results showed that a relatively short mission in space had a significant impact on both organs. The findings also indicate that immune system aberrations due to stressors associated with space travel should be included when estimating risk for pathologies such as cancer and infection and in designing appropriate countermeasures. Although this was the historic last flight of NASA’s Space Shuttle Program, exploration of space will undoubtedly continue

Spaceflight Activates Lipotoxic Pathways in Mouse Liver

Spaceflight affects numerous organ systems in the body, leading to metabolic dysfunction that may have long-term consequences. Microgravity-induced alterations in liver metabolism, particularly with respect to lipids, remain largely unexplored. Here we utilize a novel systems biology approach, combining metabolomics and transcriptomics with advanced Raman microscopy, to investigate altered hepatic lipid metabolism in mice following short duration spaceflight. Mice flown aboard Space Transportation System -135, the last Shuttle mission, lose weight but redistribute lipids, particularly to the liver. Intriguingly, spaceflight mice lose retinol from lipid droplets. Both mRNA and metabolite changes suggest the retinol loss is linked to activation of PPARα-mediated pathways and potentially to hepatic stellate cell activation, both of which may be coincident with increased bile acids and early signs of liver injury. Although the 13-day flight duration is too short for frank fibrosis to develop, the retinol loss plus changes in markers of extracellular matrix remodeling raise the concern that longer duration exposure to the space environment may result in progressive liver damage, increasing the risk for nonalcoholic fatty liver disease

Tumor response to radiotherapy is dependent on genotype-associated mechanisms in vitro and in vivo

<p>Abstract</p> <p>Background</p> <p>We have previously shown that in vitro radiosensitivity of human tumor cells segregate non-randomly into a limited number of groups. Each group associates with a specific genotype. However we have also shown that abrogation of a single gene (p21) in a human tumor cell unexpectedly sensitized xenograft tumors comprised of these cells to radiotherapy while not affecting in vitro cellular radiosensitivity. Therefore in vitro assays alone cannot predict tumor response to radiotherapy.</p> <p>In the current work, we measure in vitro radiosensitivity and in vivo response of their xenograft tumors in a series of human tumor lines that represent the range of radiosensitivity observed in human tumor cells. We also measure response of their xenograft tumors to different radiotherapy protocols. We reduce these data into a simple analytical structure that defines the relationship between tumor response and total dose based on two coefficients that are specific to tumor cell genotype, fraction size and total dose.</p> <p>Methods</p> <p>We assayed in vitro survival patterns in eight tumor cell lines that vary in cellular radiosensitivity and genotype. We also measured response of their xenograft tumors to four radiotherapy protocols: 8 × 2 Gy; 2 × 5Gy, 1 × 7.5 Gy and 1 × 15 Gy. We analyze these data to derive coefficients that describe both in vitro and in vivo responses.</p> <p>Results</p> <p>Response of xenografts comprised of human tumor cells to different radiotherapy protocols can be reduced to only two coefficients that represent 1) total cells killed as measured in vitro 2) additional response in vivo not predicted by cell killing. These coefficients segregate with specific genotypes including those most frequently observed in human tumors in the clinic. Coefficients that describe in vitro and in vivo mechanisms can predict tumor response to any radiation protocol based on tumor cell genotype, fraction-size and total dose.</p> <p>Conclusions</p> <p>We establish an analytical structure that predicts tumor response to radiotherapy based on coefficients that represent in vitro and in vivo responses. Both coefficients are dependent on tumor cell genotype and fraction-size. We identify a novel previously unreported mechanism that sensitizes tumors in vivo; this sensitization varies with tumor cell genotype and fraction size.</p

Erythroid Promoter Confines FGF2 Expression to the Marrow after Hematopoietic Stem Cell Gene Therapy and Leads to Enhanced Endosteal Bone Formation

Fibroblast growth factor-2 (FGF2) has been demonstrated to be a promising osteogenic factor for treating osteoporosis. Our earlier study shows that transplantation of mouse Sca-1+ hematopoietic stem/progenitor cells that are engineered to express a modified FGF2 leads to considerable endosteal/trabecular bone formation, but it also induces adverse effects like hypocalemia and osteomalacia. Here we report that the use of an erythroid specific promoter, β-globin, leads to a 5-fold decrease in the ratio of serum FGF2 to the FGF2 expression in the marrow cavity when compared to the use of a ubiquitous promoter spleen focus-forming virus (SFFV). The confined FGF2 expression promotes considerable trabeculae bone formation in endosteum and does not yield anemia and osteomalacia. The avoidance of anemia in the mice that received Sca1+ cells transduced with FGF2 driven by the β-globin promoter is likely due to attenuation of high-level serum FGF2-mediated stem cell mobilization observed in the SFFV-FGF2 animals. The prevention of osteomalacia is associated with substantially reduced serum Fgf23/hypophosphatemia, and less pronounced secondary hyperparathyroidism. Our improved stem cell gene therapy strategy represents one step closer to FGF2-based clinical therapy for systemic skeletal augmentation

Effect of Time and Diet on the Immune Status of Mice Challenged with Herpes-transformed Cells

Human Herpes simplex virus type 2 (HSV 2)-transformed Balb/c mouse cells (H238 cells) are malignant in Balb/c mice. The immune stratus of mice bearing H238 cell-induced tumors was studied and changes in immune response with time and with altered diet of the mice were measured. In the experiments dealing with the status of immunity with time after tumor cell injection, mice were fed Purina Laboratory Chow (P.L.C.), injected with H238 cells, and sacrificed three to eight weeks post-injection. In the diet experiments, groups of mice were fed either P.L.C. or one of six equicaloric diets, consisting of low or high levels of protein (casein or soy) and low and high levels of fat for 15 weeks before injection of the tumor cells. Non-injected and 238 (non-transformed) cell-injected mice served as the controls. The weight of each mouse was determined weekly and the amount of food consumed per cage of mice was calculated twice each week. All of these mice were sacrificed eight weeks post-injection. Size of the right thigh of all H238 cell-injected mice was measured three times per week with vernier calipers at the greatest tumor diameter. At scarified each mouse and its spleen were weighed and relative spleen weights (R.S.W) were calculated. The spleen and lymph node cell were used in a microculture lymphocyte transformation test (LTT) using both phytohemagglutinin (PHA) and lipopolysaccharide (LPS) as mitogens. Stimulation indices (S.I.) were calculated using the means counts per minute (cpm) obtained from triplicate samples. Spleen cell differential counts (S.C.D.C.) were also performed on most of the diet mice. Statistical analysis was done on the results with the use of a computerized Student\u27s T test to determine significant difference (P values). Great increases in spleen size were noted in all mice with progressive tumor, especially from three to five weeks and again from seven to eight weeks post-injection. In progressor mice the spleen cell PHA-S.I.s steadily decreased from near-normal values at three weeks (~ 51) to markedly depressed values (\u3c 10) by eight weeks. In the regressor and no-visible-tumor mice, this depression was not noted. When lymph node cells were tested, the mean PHA-S.I. was significantly elevated at four weeks post-injection, while the means obtained at all other weekly intervals were similar to the control values. The response of both spleen and lymph node cells to LPS was depressed early after tumor cell injection in mice with progressive tumor, but recovered and leveled off to normal values by eight weeks. Striking differences were observed in the incidence of tumors in the mice fed the various diets and P.L.C. All of the mice fed the high-soy protein diet and 97.5 percent of those fed P.L.C., in which the protein was mainly derived from meat, developed progressive tumors while only 55 - 60 percent of mice fed the two low-fat, casein, diets did so. Percent values for the other diet mice were intermediate. In addition. it was noted that progressor mice on the casein, low-fat diets had the longest latent period before tumor appearance and smallest tumor size at sacrifice. Progressor mice fed the casein, low-fat diets had the lowest mean R.S.W.s and the least variation from normal in the S.C.D.C. pattern, although an increase in the percent of mature granulocytes was seen in all mice with progressive tumor. Analysis of the cpm obtained during the LTT revealed that both spleen and lymph node cells of these mice were more active than those of any of the other groups in the presence or absence of PHA. A three to four-fold higher mean spleen cell PHA-S.I. was also seen in the casein-fed mice when compared to those obtained for progressor mice fed the low-fat, soy protein diets and P.L.C. The degree of depression in spleen cell PHA response in these mice was lower than that of any of the other groups. Also, these mice were the only ones to show an increase in lymph node cell responsiveness to PHA with an increasing tumor load. Humoral immunity appeared to be affected more by level rather than type of protein. Progressor mice fed the high-casein, low-fat diet had a significantly enhanced spleen cell response to IPS when compared to that of mice fed the low-casein, low-fat diet. In addition, progressor mice fed the low-soy protein, low-fat diet had a significantly higher lymph node cell response to LPS than those fed the other two soy protein diets. In mice on each of the diets and P.L.C. a range of weight gains was observed. However, body weight gain did not have a significant effect on tumor production by injection of H238 cells or on any of the parameters tested. In summary, cell-mediated immunity was increasingly depressed with time in mice with progressive H238 cell-induced tumors. Humoral immunity was depressed early after tumor cell injection, but later recovered. The results of the experiments involving diet showed than the type, in comparison to level, of protein had more effect on tumor development and immune function in the H238 tumor system, although high-soy protein did increase tumor incidence over low-soy protein. In addition, it appears that casein exerted an antitumor effect which may have been due to enhancement of cell-mediated immunity. Conversely, meat and soy protein appeared to depress cell-mediated immunity and enhance tumor development. A high level of fat was tumor-enhancing when used together with casein but tumor-inhibiting when used with soy protein

Mechanisms of Low Dose Radiation-induced T helper Cell Function

Exposure to radiation above levels normally encountered on Earth can occur during wartime, accidents such as those at Three Mile Island and Chernobyl, and detonation of “dirty bombs” by terrorists. Relatively high levels of radiation exposure can also occur in certain occupations (low-level waste sites, nuclear power plants, nuclear medicine facilities, airline industry, and space agencies). Depression or dysfunction of the highly radiosensitive cells of the immune system can lead to serious consequences, including increased risk for infections, cancer, hypersensitivity reactions, poor wound healing, and other pathologies. The focus of this research was on the T helper (Th) subset of lymphocytes that secrete cytokines (proteins), and thus control many actions and interactions of other cell types that make up what is collectively known as the immune system. The Department of Energy (DOE) Low Dose Radiation Program is concerned with mechanisms altered by exposure to high energy photons (x- and gamma-rays), protons and electrons. This study compared, for the first time, the low-dose effects of two of these radiation forms, photons and protons, on the response of Th cells, as well as other cell types with which they communicate. The research provided insights regarding gene expression patterns and capacity to secrete potent immunostimulatory and immunosuppressive cytokines, some of which are implicated in pathophysiological processes. Furthermore, the photon versus proton comparison was important not only to healthy individuals who may be exposed, but also to patients undergoing radiotherapy, since many medical centers in the United States, as well as worldwide, are now building proton accelerators. The overall hypothesis of this study was that whole-body exposure to low-dose photons (gamma-rays) will alter CD4+ Th cell function. We further proposed that exposure to low-dose proton radiation will induce a different pattern of gene and functional changes compared to photons. Over the course of this research, tissues other than spleens were archived and with funding obtained from other sources, including the Department of Radiation Medicine at the Loma Linda University Medical Center, some additional assays were performed. Furthermore, groups of additional mice were included that were pre-exposed to low-dose photons before irradiating with acute photons, protons, and simulated solar particle event (SPE) protons. Hence, the original support together with the additional funding for our research led to generation of much valuable information that was originally not anticipated. Some of the data has already resulted in published articles, manuscripts in review, and a number of presentations at scientific conferences and workshops. Difficulties in reliable and reproducible quantification of secreted cytokines using multi-plex technology delayed completion of this study for a period of time. However, final analyses of the remaining data are currently being performed and should result in additional publications and presentations in the near future. Some of the most notable conclusions, thus far, are briefly summarized below: - Distribution of leukocytes were dependent upon cell type, radiation quality, body compartment analyzed, and time after exposure. Low-dose protons tended to have less effect on numbers of major leukocyte populations and T cell subsets compared to low-dose photons. - The patterns of gene and cytokine expression in CD4+ T cells after protracted low-dose irradiation were significantly modified and highly dependent upon the total dose and time after exposure. - Patterns of gene and cytokine expression differed substantially among groups exposed to low-dose photons versus low-dose protons; differences were also noted among groups exposed to much higher doses of photons, protons, and simulated SPE protons. - Some measurements indicated that exposure to low-dose photon radiation, especially 0.01 Gy, significantly “normalized” at least some adverse effects of simulated SPE protons, thereby suggesting that this low level of radiation may induce protective mechanisms against a relatively large radiation event. - Analysis of signal transduction pathways in CD4+ T cells showed that whole-body priming with 0.01 Gy photons before exposure to simulated SPE protons significantly increased expression of p38MAPK and NF-kappaB, while JNK expression was decreased. Overall, it appears that the p38MAPK signaling pathway may be most important in inducing a radioprotective response