Differential responses to doxorubicin-induced phosphorylation and activation of Akt in human breast cancer cells

INTRODUCTION: We have shown previously that overexpression of constitutively active Akt or activation of Akt caused by constitutively active Ras or human epidermal growth factor receptor-2 (HER2) confers on breast cancer cells resistance to chemotherapy or radiotherapy. As an expanded study we here report differential responses in terms of phosphorylation and activation of Akt as a result of treatment with doxorubicin in a panel of breast cancer cell lines. METHODS: The levels of Akt phosphorylation and activity were measured by Western blot analysis with an anti-Ser473-phosphorylated Akt antibody and by in vitro Akt kinase assay using glycogen synthase kinase-3 as a substrate. RESULTS: Within 24 hours after exposure to doxorubicin, MCF7, MDA468 and T47D cells showed a drug-dose-dependent increase in the levels of phosphorylated Akt; in contrast, SKBR3 and MDA231 cells showed a decrease in the levels of phosphorylated Akt, and minimal or no changes were detected in MDA361, MDA157 and BT474 cells. The doxorubicin-induced Akt phosphorylation was correlated with increased kinase activity and was dependent on phosphoinositide 3-kinase (PI3-K). An increased baseline level of Akt was also found in MCF7 cells treated with ionizing radiation. The cellular responses to doxorubicin-induced Akt phosphorylation were potentiated after the expression of Akt upstream activators including HER2, HER3 and focal adhesion kinase. CONCLUSION: Taken together with our recent published results showing that constitutive Akt mediates resistance to chemotherapy or radiotherapy, our present data suggest that the doxorubicin-induced phosphorylation and activation of Akt might reflect a cellular defensive mechanism of cancer cells to overcome doxorubicin-induced cytotoxic effects, which further supports the current efforts of targeting PI3-K/Akt for enhancing the therapeutic responses of breast cancer cells to chemotherapy and radiotherapy

Inositides and the nucleus and inositides in the nucleus

Although there are many forms of evidence linking phosphoinositides to nuclear function, the substance of the links remains largely undefined. One link between inositide metabolism and the nucleus is suggested by the implication of inositol trisphosphate (IP3) in the process of nuclear envelope reassembly (Sullivan et al., 1993). That paper will be discussed below in its context, but this review will principally focus on another nuclear-inositide connection - a potential inositide cycle in the nucleus. It comes as something of a shock to see data that point to a phosphoinositide cycle entirely separate from the familiar one in the plasma membrane. Again contrary to expectation, the data suggest that the cycle is not in the nuclear membrane but appears to be within the nucleus. This aspect of inositide function has profound implications for the role of inositides in cell division and growth. For example, it makes us rethink the tumor-promoting actions of phorbol esters and the teratogenic effects of Li÷ that have been associated with inositide homeostasis. In this article the evidence of a nuclear inositide cycle and what is known about its control are reviewed, and the role it may play in eukaryotic cell function is discussed. For a discussion of proposed nuclear functions for protein kinase C and what little is known about nuclear Ca2÷, the reader is referred to a more comprehensive recent review (Irvine and Divecha, 1992)

Hepatocyte growth factor activates phosphoinositide 3-kinase C2 beta in renal brush-border plasma membranes

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The nuclear phosphoinositide cycle - does it play a role in nuclear Ca2+ homoeostasis?

The probable answer to this question is no. Much of the current evidence summarised elsewhere in this issue points to nuclear Ca2+ changes changing in response to cytosolic Ca2+, with little evidence for an independently controlled nuclear Ca2+ homeostasis. There are InsP3 receptors in the nuclear membrane, and it is possible that during nuclear membrane assembly the InsP3 acting on these (Sullivan and Wilson, this issue) is formed by an inositide cycle located on the assembling nuclear skeleton. But our current experimental data suggest that when the nucleus is intact, InsP3 generated by this cycle would have to exit through the nuclear pores to act on any known InsP3 receptors. Thus the nuclear inositide cycle appears more likely to serve to generate diacylglycerol to activate protein kinase C, and/or to generate inositol phosphates such as InsP2, which may have distinct intranuclear functions

Nuclear diacylglycerol is increased during cell proliferation in vivo

Highly purified nuclei were prepared from livers and kidneys of rats undergoing compensatory hepatic or renal growth, the former being predominantly by cellular proliferation, and the latter mostly by cellular enlargement. In liver, an increase in nuclear diacylglycerol (DAG) concentration occurred between 16 and 30 h, peaking at around 20 h. At the peak of nuclear DAG production a specific translocation of protein kinase C to the nucleus could be detected; no such changes occurred in kidney. There was no detectable change in whole-cell DAG levels in liver, and the increase in DAG was only measurable in nuclei freed of their nuclear membrane. Overall, these results suggest that there is a stimulation of intranuclear DAG production, possibly through the activation of an inositide cycle [Divecha, Banfic and Irvine (1991) EMBO J. 10, 3207-3214] during cell proliferation in vivo

Acetylcholine and cholecystokinin receptors functionally couple by different G‐proteins to phospholipase C in pancreatic acinar cells

We have studied the involvement of GTP‐binding proteins in the stimulation of phospholipase C from rat pancreatic acinar cells. Pretreatment of permeabilized cells with activated cholera toxin inhibited both cholecystokinin‐octapeptide (CCK‐OP) and GTPγS but not carbachol (CCh)‐induced production of inositol trisphosphate. Pertussis toxin had no effect. Neither vasoactive intestinal polypeptide, a stimulator of adenylyl cyclase, nor the cAMP‐analogue, 8‐bromo cAMP, mimicked the inhibitory effect of cholera toxin on agonist‐induced phospholipase C activation. This indicates that inhibition by cholera toxin could not be attributed to a direct interaction of cholera toxin activated Gs with phospholipase C or to an elevation of cAMP. In isolated rat pancreatic plasma membranes cholera toxin ADP‐ribosylated a 40 kDa protein, which was inhibited by CCK‐OP but not by CCh. We conclude from these data that both CCK‐ and muscarinic acetylcholine receptors functionally couple to phospholipase C by two different GTP‐binding proteins