BioProject

Caloric restriction (CR) without malnutrition is one of the most consistent strategies for increasing mean and maximal lifespan and delaying the onset of age-associated diseases. More...

Caloric restriction (CR) without malnutrition is one of the most consistent strategies for increasing mean and maximal lifespan and delaying the onset of age-associated diseases. Stress resistance is a common trait of many long-lived mutants and life-extending interventions, including CR. Indeed, better protection against heat shock and other genotoxic insults have helped explain the pro-survival properties of CR. In this study, both in vitro and in vivo responses to heat shock were investigated using two different models of CR. Murine B16F10 melanoma cells treated with serum from CR-fed rats showed lower proliferation, increased tolerance to heat shock and enhanced HSP-70 expression, compared to serum from ad libitum-fed animals. Similar effects were observed in B16F10 cells implanted subcutaneously in male C57BL/6 mice subjected to CR. Microarray analysis identified a number of genes and pathways whose expression profile were similar in both models. These results suggest that the use of an in vitro model could be a good alternative to study the mechanisms by which CR exerts its anti-tumorigenic effects. KEY WORDS: caloric restriction, heat shock, stress response, tumorigenesis, aging Overall design: In Vivo: Male C57BL/6 mice (3 month old) were single-housed in duplex caging in a room maintained at a constant temperature (20-22 °C) and humidity (30-70%) in a light:dark 12:12-h schedule, according to established animal protocols and NIH guidelines. They were fed either a standard purified mouse diet (NIH-31) ad libitum (AL; n=10) or maintained on a 40% calorie restriction regimen (CR; n=10) for the six week study. B16F10 melanoma cells (ATCC® CRL-6475™) were purchased from American Type Culture Collection (Manassas, VA); they were cultured in Dulbecco's Modified Essential Medium (DMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin (Gibco, Gaithersburg, MD) under standard cell culture conditions. One month into the study, 5 mice from each diet group were injected with 1x106 B16F10 melanoma cells in the periscapular region. Fourteen days later, animals were euthanized and tumors were excised and frozen for RNA isolation. In Vitro: B16F10 melanoma cells were incubated in media with 10% serum from either AL- or CR-fed rats, serum was obtained from overnight fasted, anesthetized 6-month-old male Fisher 344 rats. Cells were grown for 96 hours before being harvested, washed and collected for RNA isolation. The RNA was extracted using the RNeasy Mini Kit from Qiagen using standard protocols and labeled with Biotin using the standard Illumina protocol. Samples were hybed overnight to Illumina's Sentrix MouseRef-8 v1.1 Expression BeadChips. Arrays were washed, stained with Cy-3 and scanned at 0.8 micron resolution in a 500 GX Illumina Bead Array Reader. Less...