Correlations between heparan sulfate metabolism and hepatoma growth

Abstract

A rat hepatoma cell line (Gershenson et al., Science, 170:859–861, 1970) contains a dynamic steady-state pool of free heparan sulfate (HS) chains in the nucleus that increases in amount when growing cells reach confluence (Fedarko and Conrad, j. Cell Dioi, 102:587–599, 1986). In logarithmically growing cells labeled with ³⁵SO₄²⁻ steady-state levels of [³⁵SO₄]HS in the nucleus are altered by a variety of culture conditions. Rapidly dividing cells (doubling time = 18–22 h) growing under optimized conditions had steady-state levels of nuclear HS within the range of 40-50 pmol ³⁵SO₄ in nuclear HS/10⁶ cells. The steady-state levels of nuclear HS were lowered by several changes in culture conditions, including 1) additions of 1 mM p-nitrophenyl-β-D-xyloside, 0.25–0.5 mM (+)-catechin, 0.5 ng/ml transforming growth factor β, 20 ng/ml phorbol-12-myristate-13-acetate, 1 mM dibutyryl cAMP, or 1 mM inositol-2-PO₄; 2) decreased levels of D-glucose; or 3) deletions of serum, insulin, or inositol. In all cases lowering of the nuclear HS level was accompanied by an increase in the cell doubling times, suggesting a correlation in which nuclear HS levels must be optimized for maximal growth rates. When cells cultured under optimal growth conditions reached confluence, the level of nuclear HS increased threefold and the cells stopped dividing. The same culture conditions that lowered the steady-state levels of HS in the logarithmically growing cells prevented this rise in the nuclear HS as the cells reached confluence and resulted in loss of contact inhibition and overgrowth of the confluent cultures. These observations suggest a second correlation in which elevated nuclear HS levels are found when cell growth is inhibited at confluence; prevention of this rise results in continued growth. Consistent with this correlation between elevated nuclear HS and reduced growth rates, it was observed that addition of either 0.5 μg/ml hydrocortisone or 0.05 μg/ml retinoic acid to the culture medium of logarithmically growing cultures resulted in.increases in steady-state levels of nuclear HS that were accompanied by increased cell doubling times. The two agents that increased the levels of nuclear HS in logarithmically growing cultures had little effect on levels of nuclear HS in confluent cells or on contact inhibition. These results show that cells growing at maximal rates maintain nuclear HS levels within a range of 40–50 pmol SO₄ in HS/10⁶ cells and that when the steady-state levels of nuclear HS either rise above or fall below this range, the growth rates in subconfluent cultures are decreased. As log-phase cells reach confluence and stop dividing, there is a rise in the level of nuclear HS. Under growth conditions which prevent this rise, cells lose contact inhibition.