Kinetic changes of alpha B crystallin expression in neoplastic cells and syngeneic rat fibroblasts at various subculture stages

Abstract

Alpha B crystallin, a structural at variable levels, in many extraocular tissues where it plays a protective role in stress conditions. In fact, heat or toxic shocks, as well as pathological states, increase alpha B crystallin levels in many cell types. Here we show that alpha B crystallin expression is also modulated in subcultures of rat fibroblasts and Galliera sarcoma cells. Western blots analysis with anti alpha B crystallin antibodies reveals the presence of the protein in both cell populations, although the kinetic pattern of expression is different. Galliera fibroblasts constitutively express the protein up to the 70th subculture and afterwards the synthesis ceases. On the other hand, Galliera sarcoma cells do not contain alpha B crystallin in the early stages of the culture, but there is a progressive increases between the 20th and 40th cell subculture. Differences also exist concerning the intracellular distribution: alpha B crystallin is diffusely localized in the cytoplasm of fibroblasts while in sarcoma cells it localizes mainly to the perinuclear region. Alpha B crystallin is totally recovered as soluble protein in the supernatants obtained after low speed centrifugation of fibroblast homogenates, while in sarcoma cells a portion of the protein is also recovered in the insoluble pellet. Intracellular pH measurements show an alkaline cytosol in sarcoma cells compared to fibroblasts. Heat shock treatment of fibroblast subcultures constitutively expressing alpha B crystallin induces an over-expression of the protein, while in fibroblasts whose biosynthetic capacity is lost, heat shock is unable to activate the crystallin gene. Correlation between alpha B crystallin expression and proliferative rate shows that highly proliferating fibroblasts do not express alpha B crystallin, while neoplastic cells do.