In mammalian somatic-cell cycles, progression through the G1-phase restriction point and initiation of DNA replication are controlled by the ability of the retinoblastoma tumour-suppressor protein (pRb) family to regulate the E2F/DP transcription factors. Continuing transcription of E2F target genes beyond the G1/S transition is required for coordinating S-phase progression with cell division, a process driven by cyclin-B-dependent kinase and anaphase-promoting complex (APC)-mediated proteolysis. How E2F-dependent events at G1/S transition are orchestrated with cyclin B and APC activity remains unknown. Here, using an in vivo assay to measure protein stability in real time during the cell cycle, we show that repression of E2F activity or inhibition of cyclin-A-dependent kinase in S phase triggers the destruction of cyclin B1 through the re-assembly of APC, the ubiquitin ligase that is essential for mitotic cyclin proteolysis, with its activatory subunit Cdh1. Phosphorylation-deficient mutant Cdh1 or immunodepletion of cyclin A resulted in assembly of active Cdh1-APC even in S-phase cells. These results implicate an E2F-dependent, cyclin A/Cdk2-mediated phosphorylation of Cdh1 in the timely accumulation of cyclin B1 and the coordination of cell-cycle progression during the post-restriction point period.

The retinoblastoma protein (pRb)/E2F pathway regulates commitment of mammalian cells to replicate DNA. On the other hand, mitogen-stimulated cells deprived of E2F activity can still maintain physiologically relevant levels of cyclin E-dependent kinase activity and gradually enter S phase, suggesting the existence of a DNA synthesis-inducing mechanism parallel to the pRb/E2F axis. Here we show that regulatable ectopic expression of cyclin E or transcriptionally active Myc can rapidly induce DNA synthesis in U2OS-derived cell lines whose E2F activity is blocked by a constitutively active pRb (pRb⌬cdk) mutant. The effect of Myc is associated with Cdc25A phosphatase and cyclin E-CDK2 kinase activation and abolished by antagonizing Myc activity with the dominant-negative (dn) MadMyc chimera. Moreover, while abrogation of either endogenous E2F or Myc activity only delays and lowers DNA synthesis in synchronized U2OS cells or rat diploid fibroblasts, concomitant neutralization of both abolishes it. Whereas ectopic Myc and E2F1 rescue the G 1 /S delay caused by pRb⌬cdk (or dnDP1) and MadMyc, respectively, cyclin E or Cdc25A can restore DNA replication even in cells concomitantly exposed to pRb⌬cdk and MadMyc. However, coexpression of dnCDK2 neutralizes all of these rescuing effects. Finally, proper transcription of cyclin E and Cdc25A at the G 1 /S transition requires both Myc and E2F activities, and subthreshold levels of ectopic cyclin E and Cdc25A synergistically restore DNA synthesis in cells with silenced Myc and E2F activities. These results suggest that Myc controls a G 1 /Spromoting mechanism regulating cyclin E-CDK2 in parallel to the "classical" pRb/E2F pathway.The "retinoblastoma (Rb) pathway," comprising Rb protein (pRb) and its immediate upstream regulators, D-cyclins, cyclin-dependent kinases 4 and 6 (CDK4 and -6, respectively), INK4 CDK inhibitors (CKIs), and pRb-regulated E2F transcription factors, appears instrumental in regulating homeostatic tissue renewal and preventing neoplastic growth (4, 68). However, it remains to be elucidated whether this pathway represents the only physiological way to commit mammalian somatic cells to DNA replication. In particular, it is unclear whether cyclin E, a powerful inducer of cell cycle progression and DNA synthesis (31,35,37,55), is solely a primary target of the pRb/E2F axis (8,16,21,54), or whether it may also be under the control of a parallel G 1 /S-regulatory pathway. Compelling evidence exists in Drosophila and mammals for cyclin E being a pivotal downstream mediator of the Rb pathway (15,22). However, cyclin E is required and rate-limiting for G 1 /S progression in both Rb-positive and -negative cells (55). Moreover, unlike D-type cyclins, this cyclin can induce S-phase entry and DNA synthesis in mammalian cells deprived of E2F activity by expression of a constitutively active pRb mutant (pRb⌬cdk) or of a dominant-negative DP1 mutant, dnDP1 (44). These and other recent reports (29, 39) suggest that cyclin E may play multiple roles in promoting S-phase entry...

Activating mutations in the epidermal growth factor receptor gene occur as early cancer-driving clonal events in a subset of patients with non-small cell lung cancer (NSCLC) and result in increased sensitivity to EGFR-tyrosine-kinase-inhibitors (EGFR-TKIs). Despite very frequent and often prolonged clinical response to EGFR-TKIs, virtually all advanced EGFR-mutated (EGFRM+) NSCLCs inevitably acquire resistance mechanisms and progress at some point during treatment. Additionally, 20–30% of patients do not respond or respond for a very short time (<3 months) because of intrinsic resistance. While several mechanisms of acquired EGFR-TKI-resistance have been determined by analyzing tumor specimens obtained at disease progression, the factors causing intrinsic TKI-resistance are less understood. However, recent comprehensive molecular-pathological profiling of advanced EGFRM+ NSCLC at baseline has illustrated the co-existence of multiple genetic, phenotypic, and functional mechanisms that may contribute to tumor progression and cause intrinsic TKI-resistance. Several of these mechanisms have been further corroborated by preclinical experiments. Intrinsic resistance can be caused by mechanisms inherent in EGFR or by EGFR-independent processes, including genetic, phenotypic or functional tumor changes. This comprehensive review describes the identified mechanisms connected with intrinsic EGFR-TKI-resistance and differences and similarities with acquired resistance and among clinically implemented EGFR-TKIs of different generations. Additionally, the review highlights the need for extensive pre-treatment molecular profiling of advanced NSCLC for identifying inherently TKI-resistant cases and designing potential combinatorial targeted strategies to treat them.

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