A STUDY OF THE MOLECULAR CYTOLOGY OF CULTURED MONOLAYER CELLS

Abstract

Motivation of the Na'/H' exchanger in cultured monolayer cells, either directly by simple downgradient exchanges with sodium or lithium bicarbonate saline buffer, or indirectly via the phosphoinositide signal transduction pathways with ATP or inorganic sulphate, promote intracellular alkalinization. Concomitant with raised intracellular pH, is the observation of a flat-to-round (FTR) change in the morphology of anchorage dependent cells, viz. Chang liver cells and human lung fibroblasts. The rounding process is quantitated by microspectrophotometric digitization as well as by the Quantimet image analysis system. Inhibition of intracellular alkalinization with agents that block the Na'/H' antiporter, viz. incubations in sodium free medium, amiloride and quinidine (Na' /H' blockers), saxitoxin (Na' channel blocker) and staurosporine (protein kinase C inhibitor) suppress the rounding responses as well. A linkage between intracellular alkalinization and cell rounding seems suggested. The levels of phosphoinositide second messengers, diacylglycerol and inositol trisphosphate, are shown to be elevated concurrently with pH1 upshifts and cell rounding in sulphate induction. In addition, raised cytosolic calcium in ATP incubated Chang cells, which have been preloaded with Fluo 3 dye, is demonstrated with the argon laser confocal microscope. Both intracellular alkalinization and the antiporter mediated rounding phenomenon are shown to be dissociable from DNA synthesis, a prerequisite in cell proliferation, using the DNA inhibitor, genistein. Ultrastructural studies of the rounding process, reveal a distinctive and novel, large channel and non-clathrin coated endocytosis. As the cells round up, there is a dramatic reduction in the cell profile area and perimeter due to the internalization of the plasma membrane. Extracellular matrix of confluent Chang liver and human keloid fibroblast cultures, which are stained by fluorescein-conjugated moroclonal anti-fibronectin, disappeared upon rounding. Large macromolecules such as fluoresceinated dextran of 2 million molecular weight are easily internalized as evidenced by quantitative spectrofluorimetric evaluation. This new endocytotic pathway provides a new way of loading cells with impermeant macromolecules. The other cytological manifestations of the rounding process include: (a) enlarged rough endoplasmic reticulum (ER) channels, loss of ribosomal granules with transformation into smooth ER and the subsequent conversion into lysosomes, and (b) microvillus processes that anchor the rounded cell to the substratum. The rounded cell is also observed to share certain common characteristics with the mitotic cell. Distinctive neutral red dye uptake is observed in cancer mitotic cells, viz. A431 human skin epidermoid carcinoma cells, mouse Cloudman malignant melanoma cells, human oral carcinoma cells and rat hepatoma cells. This characteristic cannot be simulated by antiporter mediated endocytosis in non-tumorigenic, interphase Chang liver cells but can be mimicked by incubation with epidermal growth factor as demonstrated in both Chang liver and A431 carcinoma cells. A causal relationship with tyrosyl phosphorylation seems suggested.