In the present study, we demonstrate that Ca 2 þ -induced growth inhibition and induction of differentiation in a line of human colon carcinoma cells (CBS) is dependent on mitogen-activated protein (MAP) kinase signaling and is associated with upregulation of extracellular calcium-sensing receptor (CaSR) expression. When CBS cells were grown in Ca 2 þ -free medium and then switched to medium supplemented with 1.4 mM Ca 2 þ , proliferation was reduced and morphologic features of differentiation were expressed. E-cadherin, which was minimally expressed in nonsupplemented medium, was rapidly induced in response to Ca 2 þ stimulation. Sustained activation of the extracellular signal-regulated kinase (ERK) occured in Ca 2 þ -supplemented medium. When an inhibitor of ERK activation (10 mM U0126) was included in the Ca 2 þ -supplemented culture medium, ERK-activation did not occur. Concomitantly, E-cadherin was not induced, cell proliferation remained high and differentiation was not observed. The same level of Ca 2 þ supplementation that induced MAP kinase activation also stimulated CaSR upregulation in CBS cells. A clonal isolate of the CBS line that did not upregulate CaSR expression in response to extracellular Ca 2 þ was isolated from the parent cells. This isolate failed to produce E-cadherin or undergo growth inhibition/induction of differentiation when exposed to Ca 2 þ in the culture medium. However, ERK-activation occurred as efficiently in this isolate as in parent CBS cells or in a cloned isolate that underwent growth reduction and differentiation in response to Ca 2 þ stimulation. Together, these data indicate that CaSR upregulation and MAP kinase signalling are both intermediates in the control of colon carcinoma cell growth and differentiation. They appear to function, at least in part, independently of one another.

a b s t r a c t KP1019 comprises a class of ruthenium compounds having promising anticancer activity. Here, we investigated the molecular targets of KP1019 using Saccharomyces cerevisiae as a model organism. Our results revealed that in the absence of the N-terminal tail of histone H3, the growth inhibitory effect of KP1019 was markedly enhanced. Furthermore, H3K56A or rtt109D mutants exhibit hypersensitivity for KP1019. Moreover, KP1019 evicts histones from the mononucleosome and interacts specifically with histone H3. We have also shown that KP1019 treatment causes induction of Ribonucleotide Reductase (RNR) genes and degradation of Sml1p. Our results also suggest that DNA damage induced by KP1019 is primarily repaired through double-strand break repair (DSBR). In summary, KP1019 targets histone proteins, with important consequences for DNA damage responses and epigenetics.

The calcium-sensing receptor (CaSR) is a G-coupled protein expressed in renal juxtaglomerular (JG) cells. Its activation stimulates calcium-mediated decreases in cAMP content and inhibits renin release. The postreceptor pathway for the CaSR in JG cells is unknown. In parathyroids, CaSR acts through Gq and/or Gi. Activation of Gq stimulates phospholipase C (PLC), and inositol 1,4,5-trisphosphate (IP3), releasing calcium from intracellular stores. Gi stimulation inhibits cAMP formation. In afferent arterioles, the ryanodine receptor (RyR) enhances release of stored calcium. We hypothesized JG cell CaSR activation inhibits renin via the PLC/IP3 and also RyR activation, increasing intracellular calcium, suppressing cAMP formation, and inhibiting renin release. Renin release from primary cultures of isolated mouse JG cells (n ϭ 10) was measured. The CaSR agonist cinacalcet decreased renin release 56 Ϯ 7% of control (P Ͻ 0.001), while the PLC inhibitor U73122 reversed cinacalcet inhibition of renin (104 Ϯ 11% of control). The IP3 inhibitor 2-APB also reversed inhibition of renin from 56 Ϯ 6 to 104 Ϯ 11% of control (P Ͻ 0.001). JG cells were positively labeled for RyR, and blocking RyR reversed CaSR-mediated inhibition of renin from 61 Ϯ 8 to 118 Ϯ 22% of control (P Ͻ 0.01). Combining inhibition of IP3 and RyR was not additive. Gi inhibition with pertussis toxin plus cinacalcet did not reverse renin inhibition (65 Ϯ 12 to 41 Ϯ 8% of control, P Ͻ 0.001). We conclude stimulating JG cell CaSR activates G q, initiating the PLC/IP3 pathway, activating RyR, increasing intracellular calcium, and resulting in calcium-mediated renin inhibition. calcium; renin; calcium-sensing receptor; inositol-3 phosphate; phospholipase C; protein kinase A; ryanodine receptor THE CALCIUM-SENSING RECEPTOR (CaSR) is a G protein-coupled 7-transmembrane receptor (33) found in the parathyroid gland, blood vessels, and kidney (18). In the parathyroid gland where CaSR was first described, high extracellular calcium results in decreased parathyroid hormone secretion (21). This is mediated by calcium activation of the CaSR, stimulating the G proteins G q and/or G i . In the parathyroid, activation of G q stimulates phospholipase C (PLC), producing diacylglycerol and inositol 1,4,5-trisphosphate (IP 3 ), the latter of which releases calcium from intracellular stores bound in the endoplasmic reticulum (ER) (48). G i stimulation leads to the inhibition of cAMP formation (16,48). Either pathway results in suppressing parathyroid hormone release.In the secretory juxtaglomerular (JG) cells, like the parathyroids, increased calcium results in the suppression of renin secretion (21,43,44). We have previously reported, both in vitro (44) and in vivo (6), that the JG cells contain CaSR, and that increased media calcium or activation of the CaSR with a calcimimetic (6, 43, 44) leads to suppression of renin secretion. JG cells synthesize, store, and release renin, the rate-limiting enzyme involved in the formation of angiotensin II (9). The cyclic nucleotide cAMP ...

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