Outcome of treatment of human HeLa cervical cancer cells with roscovitine strongly depends on the dosage and cell cycle status prior to the treatment

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 G₂/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 G₁/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 G₁ 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 G₁ block after serum starvation, but respond with a slight increase of the ratio of G₁ population. Exposure of G₁‐enriched HeLa cells to ROSC after re‐feeding does not block their cell‐cycle progression at G₁‐phase, but increases the ratio of S‐ and G₂‐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 G₁‐phase. These results show that inhibition of CDKs by ROSC in cells lacking the G₁/S restriction checkpoint has different outcomes depending on the cell‐cycle status prior to the onset of treatment. J. Cell. Biochem. 106: 937–955, 2009. © 2009 Wiley‐Liss, Inc.     

Why Callosobruchus rhodesianus causes limited damage during storage of cowpea seeds in a tropical humid zone in Togo

The sympatric bruchids Callosobruchus rhodesianus (Pic) and Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) colonize cultures of cowpea, Vigna unguiculata (Walp.) (Papilionaceae), in Togo at the end of the rainy season. Seeds containing larvae of the two bruchid species were introduced into the storage systems. Callosobruchus rhodesianus adults emerged from the seeds at the beginning of storage, but this species disappeared quickly and C. maculatus became dominant. Analysis of the reproduction of females collected at the beginning of storage made it possible to distinguish between four groups of females differing in their reproductive activity. Three groups were sexually active but they differed in their fecundity and the duration of the latency period before the first oviposition phase. The fourth group of females was in reproductive diapause. In interspecific competition, the presence of C. maculatus larvae reduced the survival chances of C. rhodesianus and therefore is disadvantageous for this species. The main factors explaining the rapid decrease of the C. rhodesianus populations under storage conditions were the variability of female reproductive activity with the emergence of insects in reproductive diapause and the significant larval mortality in interspecific competition with C. maculatus.     

TSPO ligand PK11195 and MAPK inhibitor UO126 modulate TSPO expression in melanoma cells

TSPO (translocator protein) is an intracellular protein involved in the regulation of cell proliferation, differentiation, and apoptosis. However, the mechanisms of TPSO regulation are poorly understood. We have studied the action of MAPK inhibitor UO126 and TSPO specific ligand PK11195 on the TSPO expression level in melanoma cells. It was found that, in nanomolar concentrations, PK11195 increased the TPSO level in skin melanoma cells. This result was confirmed with UO126 inhibitor by real-time PCR and immunocytochemistry. A combination of PK11195 and UO126 activated apoptotic death of melanoma cells. The data obtained may be used to regulate the functional activity of TPSO-mediated processes.     

Reconstitution of human MCF-7 breast cancer cells with caspase-3 does not sensitize them to action of CDK inhibitors

Human MCF-7 breast cancer cells are resistant to pro-apoptotic stimuli due to caspase-3 inactivation. On the other hand, they should be sensitive to agents like selective pharmacological inhibitors of cyclin-dependent kinases (CDKs) that (re)activate p53 tumor suppressor protein because they harbor intact p53 pathways. In this study we examined whether reconstitution of caspase-3 in MCF-7 cells sensitizes them to inhibitors of CDKs, by analyzing the effects of roscovitine (ROSC) and olomoucine (OLO), two closely related selective pharmacological CDK inhibitors, on both mother MCF-7 cells and a secondary mutant line, MCF-7.3.28 that stably expresses human caspase-3. The results show that ROSC is, as expected, much more potent than OLO. Surprisingly; however, ROSC and OLO reduced proliferation of parental MCF-7 cells more strongly than caspase-3-proficient counterparts. Both inhibitors arrest human breast cancer cells at the G(2)-phase of the cell cycle. Analysis of cell-cycle regulators by immunoblotting revealed that ROSC strongly induces p53 protein activity by inducing its phosphorylation at Ser46 in the MCF-7 cells lacking caspase-3, but not in caspase-3-proficient cells. Furthermore, reconstitution of caspase-3 in MCF-7 cells neither elevates the mitochondrial apoptosis rate nor significantly increases caspases activity upon ROSC treatment. However, the stabilization of p53 in response to DNA damaging agents is the same in both caspase negative and positive MCF-7 cells. Cytotoxic agents induce caspase-3-dependent apoptosis in caspase-3-proficient cells. These results indicate that reconstitution of MCF-7 cancer cells with caspase-3 sensitize them to the action of DNA damaging agents but not to ATP-like pharmacological inhibitors of CDKs.     

Characterization of the antibodies to p62 nucleoporin in primary biliary cirrhosis using human recombinant antigen

Reactivity of sera from patients with primary biliary cirrhosis (PBC) with a 60 kDa component of nuclear pore complexes (NPCs), purified by affinity chromatography on wheat-germ agglutinin (WGA)-Sepharose, was previously detected. Recently, clinical significance of the anti-NPC antibodies in PBC became evident. In the light of recent reports, indicating the correlation of the anti-NPC antibodies with severity and progression of the disease, the characterization of the reactive antigens is becoming essential in the clinical management of patients with PBC. Since accurate autoantibody detection represents one of the fundamental requirements for a reliable testing, we have generated a human recombinant p62 protein and validated an immunoprecipitation assay for the detection of anti-p62. We also demonstrated that the generated human recombinant p62 nucleoporin was modified by N-acetylglucosamine residues. More than 50% of tested PBC sera precipitated (35)S-radioactively labeled p62 recombinant nucleoporin and 40% recognized this recombinant antigen by immunoblotting. We compared the reactivity of PBC sera with rat and human nucleoporin. The incidence of anti-p62 nucleoporin positive PBC sera increased by 15% when human recombinant antigen was used. The titer of autoantibodies in p62-positive PBC samples strongly varied. Preadsorption of the PBC sera with p62 recombinant protein completely abolished their reactivity with the antigen. In conclusion, this study unequivocally proves that autoantibodies reacting with the 60 kDa component of NPCs target p62 nucleoporin and, more importantly, provide a better antigen source for future evaluations of the clinical role of anti-p62 in PBC.     

In vivo and in vitro phosphorylation of membrane and soluble forms of soybean nodule sucrose synthase

Sucrose synthase (SS) is a known phosphoserine (SerP)-containing enzyme in a variety of plant "sink" organs, including legume root nodules, where it is phosphorylated primarily at Ser-11. Using immunofluorescence confocal microscopy, we documented that part of the total SS (nodulin-100) pool in mature soybean (Glycine max) nodules is apparently associated with the plasma membrane in situ, and we report that this association is very "tight," as evidenced by a variety of chemical and enzymatic pretreatments of the isolated microsomal fraction. To investigate the in situ and in planta phosphorylation state of the membrane (m) and soluble (s) forms of nodule SS, three complementary approaches were used. First, excised nodules were radiolabeled in situ with [(32)P]Pi for subsequent analysis of phosphorylated m- and s-SS; second, immunopurified s- and m-SS were used as substrate in "on-bead" assays of phosphorylation by nodule Ca(2+)-dependent protein kinase; and third, SS-Ser-11(P) phosphopeptide-specific antibodies were developed and used. The collective results provide convincing evidence that microsomal nodulin-100 is phosphorylated in mature nodules, and that it is hypophosphorylated relative to s-SS (on an equivalent SS protein basis) in attached, unstressed nodules. Moreover, the immunological data and related phosphopeptide mapping analyses indicate that a homologous N-terminal seryl-phosphorylation domain and site reside in microsomal nodulin-100. We also observed that mild, short-term inorganic nitrogen and salt stresses have a significant negative impact on the content and N-terminal phosphorylation state of nodule m- and s-SS, with the former being the more sensitive of the two SS forms.     

Sucrose Synthase in Legume Nodules Is Essential for Nitrogen Fixation

The role of sucrose synthase (SS) in the fixation of N was examined in the rug4 mutant of pea (Pisum sativum L.) plants in which SS activity was severely reduced. When dependent on nodules for their N supply, the mutant plants were not viable and appeared to be incapable of effective N fixation, although nodule formation was essentially normal. In fact, N and C resources invested in nodules were much greater in mutant plants than in the wild-type (WT) plants. Low SS activity in nodules (present at only 10% of WT levels) resulted in lower amounts of total soluble protein and leghemoglobin and lower activities of several enzymes compared with WT nodules. Alkaline invertase activity was not increased to compensate for reduced SS activity. Leghemoglobin was present at less than 20% of WT values, so O₂ flux may have been compromised. The two components of nitrogenase were present at normal levels in mutant nodules. However, only a trace of nitrogenase activity was detected in intact plants and none was found in isolated bacteroids. The results are discussed in relation to the role of SS in the provision of C substrates for N fixation and in the development of functional nodules.     

Whether to target single or multiple CDKs for therapy? That is the question

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. J. Cell. Physiol. 226: 341–349, 2011. © 2010 Wiley‐Liss, Inc.