Mutations of the epidermal growth factor receptor (EGFR) selectively activate Akt and signal transducer and activator of transcription (STAT) pathways that are important in lung cancer cell survival. Src family kinases can cooperate with receptor tyrosine kinases to signal through downstream molecules, such as phosphatidylinositol 3-kinase/PTEN/Akt and STATs. Based on the importance of EGFR signaling in lung cancer, the known cooperation between EGFR and Src proteins, and evidence of elevated Src activity in human lung cancers, we evaluated the effectiveness of a novel orally bioavailable Src inhibitor dasatinib (BMS-324825) in lung cancer cell lines with defined EGFR status. Here, we show that cell fate (death versus growth arrest) in lung cancer cells exposed to dasatinib is dependent on EGFR status. In cells with EGFR mutation that are dependent on EGFR for survival, dasatinib reduces cell viability through the induction of apoptosis while having minimal apoptotic effect on cell lines with wild-type (WT) EGFR. The induction of apoptosis in these EGFR-mutant cell lines corresponds to down-regulation of activated Akt and STAT3 survival proteins. In cell lines with WT or resistant EGFR mutation that are not sensitive to EGFR inhibition, dasatinib induces a G 1 cell cycle arrest with associated changes in cyclin D and p27 proteins, inhibits activated FAK, and prevents tumor cell invasion. Our results show that dasatinib could be effective therapy for patients with lung cancers through disruption of cell growth, survival, and tumor invasion. Our results suggest EGFR status is important in deciding cell fate in response to dasatinib.

Our studies address questions pertaining to the regulation of D cyclin-cdk4 activity, and the following results were obtained. Conditions that increased the abundance of the D cyclins also increased the abundance of enzymatically active D cyclin-cdk4 complexes in mouse embryo fibroblasts (MEFs) lacking both p27 Kip1 The cyclin-dependent kinases (CDKs) mediate the passage of cells through the cell cycle (13). Two families of proteins regulate CDK activity: the cyclins, which are required for activity, and the CDK inhibitors (CKIs). Two CKIs, p27Kip1 and p21Cip1 , associate with complexes containing cyclins D, E, and A and their CDK partners. The D cyclins (D1, D2, and D3) increase in abundance and combine with cdk4 or cdk6 in midto late G 1 , and the resultant complexes phosphorylate the retinoblastoma protein (Rb) and sequester p27Kip1 and p21Cip1 . Sequestration of p27 Kip1 and p21 Cip1 prevents their interaction with, and the consequent inactivation of, cyclin E-cdk2 and cyclin A-cdk2. When active, these complexes further phosphorylate Rb and elicit additional events required for the initiation and execution of S phase.Although p27 Kip1 and p21 Cip1 clearly inhibit cdk2 activity, their effects on cdk4 activity are unresolved. Some studies suggest that p27Kip1 and p21 Cip1 function as obligate assembly factors for D cyclin-cdk4 complexes (4) and that D cyclin-cdk4 complexes containing a single p27Kip1 or p21 Cip1 are active (2, 9, 19). Other studies argue that D cyclin-cdk4 complexes containing p27Kip1 or p21 Cip1 are always inactive. For example, we found that antibody to p27Kip1 or p21 Cip1 coprecipitated D cyclin-cdk4 complexes from extracts of growing fibroblasts but did not remove cdk4 activity (1). Moreover, as reported by us and others, p27Kip1 inhibited cdk4 activity in vitro over a wide range of concentrations (1, 9). We suggest that D cyclin-cdk4 complexes fulfill their enzymatic and sequestration obligations by devoting different portions of the D cyclin-cdk4 pool to each function (1). Inhibitory effects of p27Kip1 and p21 Cip1 on cdk4 activity indicate that these CKIs cannot be required for D cyclin-cdk4 complex formation. Consistent with this premise, we found that D cyclin-cdk4 complexes were present in mouse embryo fibroblasts (MEFs) lacking both p27Kip1 and p21 Cip1 (p27/ p21 Ϫ/Ϫ ), although in smaller amounts than in wild-type MEFs (1). We suggest that the low abundance of D cyclin-cdk4 complexes in p27/p21 Ϫ/Ϫ MEFs reflects the inherent instability of binary D cyclin-cdk4 complexes (9) rather than the need for p27 Kip1 or p21 Cip1 for complex assembly. As described in a previous report (1), most of the D cyclincdk4 complexes in asynchronously cycling wild-type MEFs contain p27Kip1 or p21 Cip1 , whereas the remaining complexes, although almost undetectable, account for all of the D cyclinassociated activity. Thus, in both wild-type and p27/p21 Ϫ/Ϫ MEFs, mechanisms limit the extent and duration of D cyclincdk4 activation. In the presence of p27Kip1 and p21 Cip1 , inactive ternary complexes pr...

A type I topoisomerase has been purified to homogeneity from Mycobacterium smegmatis. It is the largest single subunit enzyme of this class having molecular mass of 110 kDa. The enzyme is Mg 2؉ dependent and can relax negatively supercoiled DNA, catenate, and knot single-stranded DNA, thus having typical properties of type I topoisomerases. Furthermore, the enzyme makes single-stranded nicks and the 5-phosphoryl end of the nicked DNA gets covalently linked with a tyrosine residue of the enzyme. However, M. smegmatis enzyme shows some distinctive features from the prototype Escherichia coli topoisomerase I. The enzyme is relatively stable at higher temperatures and not inhibited by spermidine. It apparently does not contain any bound Zn 2؉ and on modification of cysteine residues retains the activity, suggesting the absence of the zincfinger motif in DNA binding. Partially purified Mycobacterium tuberculosis topoisomerase I exhibits very similar properties with respect to size, stability, and reaction characteristics. Sequence comparison of topoisomerase I from E. coli and M. tuberculosis shows the absence of zinc-finger motifs in mycobacterial enzyme. Using a two-substrate assay system, we demonstrate that the enzyme acts processively at low ionic strength and switches over to distributive mode at high Mg 2؉ concentration. Significantly, the enzyme activity is stimulated by single strand DNA-binding protein. There is a potential to exploit the characteristics of the enzyme to develop it as a molecular target against mycobacterial infections.

Our studies examined the effects of p27 kip1 and p21 cip1 on the assembly and activity of cyclin D3-cdk4 complexes and determined the composition of the cyclin D3 pool in cells containing and lacking these cyclin-dependent kinase inhibitors. We found that catalytically active cyclin D3-cdk4 complexes were present in fibroblasts derived from p27 kip1 -p21 cip1-null mice and that immunodepletion of extracts of wild-type cells with antibody to p27 kip1 and/or p21 cip1 removed cyclin D3 protein but not cyclin D3-associated activity. Similar results were observed in experiments assaying cyclin D1-cdk4 activity. Data obtained using mixed cell extracts demonstrated that p27 kip1 interacted with cyclin D3-cdk4 complexes in vitro and that this interaction was paralleled by a loss of cyclin D3-cdk4 activity. In p27 kip1 -p21cip1 -deficient cells, the cyclin D3 pool consisted primarily of cyclin D3 monomers, whereas in wild-type cells, the majority of cyclin D3 molecules were complexed to cdk4 and either p27 kip1 or p21 cip1 or were monomeric. We conclude that neither p27 kip1 nor p21 cip1 is required for the formation of cyclin D3-cdk4 complexes and that cyclin D3-cdk4 complexes containing p27 kip1 or p21 cip1 are inactive. We suggest that only a minor portion of the total cyclin D3 pool accounts for all of the cyclin D3-cdk4 activity in the cell regardless of whether the cell contains p27 kip1 and p21 cip1.Cell cycle progression is regulated by an ordered sequence of events that includes the activation of the cyclin-dependent kinases (cdk's) (37, 43). Activation of the cdk's requires both their association with cyclins, whose levels fluctuate during the cell cycle, and their phosphorylation at specific threonine residues by cdk-activating kinase, a constitutively expressed enzyme (46). cdk's also interact with a group of proteins collectively termed cdk inhibitors (CKIs); CKI levels, like cyclin levels, vary during the cell cycle and thus contribute to the timing of cdk activation (44,45). Traverse of G 0 /G 1 and entry into S phase is controlled by the sequential activation of complexes containing the D cyclins and cdk4 or cdk6, cyclin E and cdk2, and cyclin A and cdk2. This series of events is initiated by mitogen-induced increases in the expression of the D cyclins (D1, D2, and D3) and the formation of active cyclin D-cdk4 (or cdk6) complexes in mid-G 1 (30,50). D cyclin-containing complexes phosphorylate the antioncogene Rb, as do cyclin E-cdk2 complexes, which become active in late G 1 due, at least in part, to decreases in CKI levels (16,20,22,29,49). When phosphorylated by these kinases, Rb no longer represses the activity of the E2F transcription factors, and a variety of E2F target genes, including those encoding cyclin E, cyclin A, and several DNA replication enzymes, are expressed (6,13,48). At this point, cells pass through the restriction point in late G 1 and, in a manner dependent on cyclin E-cdk2 and cyclin A-cdk2 activity, enter and traverse S phase (36).Two classes of CKIs have been defined: the INK pro...