Complexes consisting of cyclin-dependent kinases (CDKs) and their regulatory subunits (the cyclins) control the progression of normal mammalian cells through the cell cycle. However, during malignant transformation this regulatory apparatus malfunctions, allowing cells to undergo unchecked proliferation. In many cases, the high mitotic potential of malignant cells is due to the constitutive activation of CDK-cyclin complexes, facilitated by the inactivation of cellular CDK inhibitors, such as p16(INK4A) or p27(Kip1), and the loss of functional tumor suppressors, such as the p53 and pRb proteins. It has recently been suggested that pharmacological intervention based on remedying the deficiency or loss of activity of these negative regulators of the cell cycle could be a very effective therapeutic option in the treatment of cancer. Multiple CDK inhibitors have been synthesized over the last two decades, spanning at least five classes of compounds. While these inhibitors can be classified on the basis of their chemical structure, it may be more interesting to categorize them according to their pharmacological nature, as broad-spectrum unspecific, pan-specific, or very selective antagonists. This review offers a critical assessment of the advantages and disadvantages of both pan-specific and highly selective CDK inhibitors in therapy.

Background: Epidermal growth factor receptor (EGFR) and mammalian target of rapamycin (mTOR) are crucial targets in cancer therapy. Combined inhibition of both targets yielded synergistic effects in vitro and in vivo in several cancer entities. However, the impact of EGFR and mTOR expression and combined inhibition in neuroendocrine lung tumors other than small-cell lung cancer remains unclear. Material and Methods: Expression and activation of EGFR/AKT/mTOR pathway constituents were investigated in typical and atypical bronchial carcinoid (AC) tumors and large-cell neuroendocrine lung carcinomas (LCNEC) by immunohistochemistry in 110 tumor samples, and correlated with clinicopathological parameters and patient survival. Cytotoxicity of mTOR inhibitor everolimus and EGFR inhibitor erlotinib alone and in combination was assessed using growth inhibition assay in NCI-H720 AC and SHP-77 LCNEC cells. Cell cycle phase distribution was determined by FACS. Apoptosis-associated activation of caspase-3/7 was measured by Caspase-Glo® assay. Activity status of EGFR and mTOR pathway components was analyzed by immunoblotting. Results: Activation of the EGFR/AKT/mTOR axis could be demonstrated in all entities and was significantly increased in higher grade tumors. Neoadjuvant chemotherapy correlated significantly with p-AKT expression and p-ERK loss. Erlotinib combined with everolimus exerted synergistic combination effects in AC and LCNEC cells by induction of apoptosis, while cell cycle phase distribution remained unaffected. These effects could be explained by synergistic downregulation of phospho-mTOR, phospho-p70S6 kinase and phospho-AKT expression by everolimus and erlotinib. Conclusions: Our study indicates that EGFR and mTOR are clinically important targets in bronchial neuroendocrine tumors, and further in vivo and clinical exploration of combined inhibition is warranted.

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Exposure of asynchronously growing human HeLa cervical carcinoma cells to roscovitine (ROSC), a selective cyclin-dependent kinases (CDKs) inhibitor, arrests their progression at the transition between G(2)/M and/or induces apoptosis. The outcome depends on the ROSC concentration. At higher dose ROSC represses HPV-encoded E7 oncoprotein and initiates caspase-dependent apoptosis. Inhibition of the site-specific phosphorylation of survivin and Bad, occurring at high-dose ROSC treatment, precedes the onset of apoptosis and seems to be a prerequisite for cell death. Considering the fact that in HeLa cells the G(1)/S restriction checkpoint is abolished by E7, we addressed the question whether the inhibition of CDKs by pharmacological inhibitors in synchronized cells would be able to block the cell-cycle in G(1) phase. For this purpose, we attempted to synchronize cells by serum withdrawal or by blocking of the mitotic apparatus using nocodazole. Unlike human MCF-7 cells, HeLa cells do not undergo G(1) block after serum starvation, but respond with a slight increase of the ratio of G(1) population. Exposure of G(1)-enriched HeLa cells to ROSC after re-feeding does not block their cell-cycle progression at G(1)-phase, but increases the ratio of S- and G(2)-phase, thereby mimicking the effect on asynchronously growing cells. A quite different impact is observed after treatment of HeLa cells released from mitotic block. ROSC prevents their cell cycle progression and cells transiently accumulate in G(1)-phase. These results show that inhibition of CDKs by ROSC in cells lacking the G(1)/S restriction checkpoint has different outcomes depending on the cell-cycle status prior to the onset of treatment.

Roscovitine (ROSC), a selective cyclin-dependent kinase (CDK) inhibitor, arrests human estrogen receptor-α (ER-α) positive MCF-7 breast cancer cells in the G(2) phase of the cell cycle and concomitantly induces apoptosis via a p53-dependent pathway. The effect of ROSC is markedly diminished in MCF-7 cells maintained in the presence of estrogen-mimicking compounds. Therefore, we decided to examine whether ROSC has any effect on the functional status of the ER-α transcription factor. Exposure of MCF-7 cells to ROSC abolished the activating phosphorylation of CDK2 and CDK7 in a concentration and time-dependent manner. This inhibition of site-specific modification of CDK7 at Ser164/170 prevented phosphorylation of RNA polymerase II and reduced basal phosphorylation of ER-α at Ser118 in non-stimulated MCF-7 cells (resulting in its down-regulation). In MCF-7 cells, estrogen induced strong phosphorylation of ER-α at Ser118 but not at Ser104/Ser106. ROSC prevented this estrogen-promoted activating modification of ER-α. Furthermore, we sought to determine whether the activity of ROSC could be enhanced by combining it with an anti-estrogen. Tamoxifen (TAM), a selective estrogen receptor modulator (SERM), affected breast cancer cell lines irrespective of their ER status. In combination with ROSC, however, it had a different impact, enhancing G(1) or G(2) arrest. Our results indicate that ROSC prevents the activating phosphorylation of ER-α and that its mode of action is strongly dependent on the cellular context. Furthermore, our data show that ROSC can be combined with anti-estrogen therapy. The inhibitory effect of TAM on ER-negative cancer cells indicates that SERMs crosstalk with other steroid hormone receptors.

Antibodies against nuclear components (ANAs) occur in sera of approximately 50% of patients with primary biliary cirrhosis (PBC). By indirect immunofluorescence (IIF) ANA-positive PBC sera generate most frequently, homogeneous, speckled, centromere, and rim-like staining patterns. A perinuclear staining pattern is indicative for the reactivity of the sera with the components of the nuclear envelope. A substantial subset of PBC patients develops antibodies against constituents of the nuclear pore complexes (NPCs). These autoantibodies target two major autoantigens: gp210 glycoprotein and p62 kDa nucleoporin. Originally, a strong reaction of PBC with a 60 kDa protein of NPCs that was affinity purified on wheat-germ agglutinin (WGA)-Sepharose was described. Recently, using human recombinant p62 nucleoporin the identity of the reactivity was confirmed. In this work we compared by immunoprecipitation the reactivity of 20 PBC sera with the two recombinant autoantigens of the NPCs. Two out of 20 (10%) PBC sera precipitated recombinant gp210 glycoprotein and 11 out of 20 (55%) PBC sera reacted with p62 nucleoporin. These results evidence that anti-p62 antibodies occur more frequently than the autoantibodies against gp210 glycoprotein. Considering the recently reported clinical significance of ANAs in PBC, the prognostic value of the anti-NPC antibodies and their correlation with severity and progression of the disease is under evaluation.

Chronic lymphocytic leukemia (CLL) is characterized by the accumulation of malignant, apoptosis-resistant B CD19(+)/CD5(+) cells. Populations of CLL cells are heterogeneous and consist primarily of quiescent cells with a minor subset of dividing cells. In this study the efficacy of a first-line in vivo therapy was compared with treatment by R-roscovitine (ROSC) alone or by purine analogues (cladribine and fludarabine) combined with maphosphamide for 14 CLL patients under ex vivo conditions. ROSC induced the highest reduction in numbers of living B-cells, coinciding with an increased rate of apoptosis. After 24 h the percentage of apoptotic cells in ROSC-treated cultures was markedly higher than in untreated controls. ROSC also induced strong activation of the apoptosome and effector caspases in CLL cells. During progression of apoptosis the plasma membrane became permeable, resulting in the release of activated caspases into the culture medium. Leukemic cells were more sensitive to ROSC than normal mononuclear cells. Treatment with ROSC did not affect the activating phosphorylation of CDK2 or CDK1. However, ROSC decreased phosphorylation of survivin, CDK7, and RNA-Pol II, resulting in inhibition of transcription elongation and subsequent down-regulation of levels of anti-apoptotic factors, thereby facilitating apoptosis. Unlike ROSC, two other purine analogues barely affected the cellular levels of anti-apoptotic proteins and more weakly activated effector caspases. In addition, the efficacies of in vivo and ex vivo therapies were found to be correlated. Marked between-patient differences in expression patterns of apoptosis-regulating factors in CLL cells were observed, explaining the variations in patients' sensitivity to therapy.

Increased expression and activity of proteins driving cell cycle progression as well as inactivation of endogenous inhibitors of cyclin-dependent kinases (CDKs) enhance the proliferative potential of cells. Escape of cells during malignant transformation from the proper cell cycle control rendering them independent from growth factors provides rationale for therapeutic targeting of CDKs. Exposure of rapidly growing human MCF-7 breast cancer and HeLa cervix cancer cells to roscovitine (ROSC), a selective inhibitor of CDKs, inhibits their proliferation by induction of cell cycle arrest and/or apoptosis. The outcome strongly depends on the intrinsic traits of the tumor cells, on their cell cycle status prior to the onset of treatment and also on ROSC concentration. At lower dose ROSC primarily inhibits the cell cycle-related CDKs resulting in a strong cell cycle arrest. Interestingly, ROSC arrests asynchronously growing cells at the G(2)/M transition irrespective of the status of their restriction checkpoint. However, the exposure of cancer cells synchronized after serum starvation in the late G(1) phase results in a transient G(1) arrest only in cells displaying the intact G(1)/S checkpoint. At higher dosage ROSC triggers apoptosis. In HeLa cells inhibition of the activity of CDK7 and, in consequence, that of RNA polymerase II is a major event that facilitates the initiation of caspase-dependent apoptosis. In contrast, in the caspase-3-deficient MCF-7 breast cancer cells ROSC induces apoptosis by a p53-dependent pathway. HIPK2-mediated activation of the p53 transcription factor by phosphorylation at Ser46 results in upregulation of p53AIP1 protein. This protein after de novo synthesis and translocation into the mitochondria promotes depolarization of the mitochondrial membrane.