BACKGROUND
phosphorylation is the most common post-translational protein modification that regulates a wide spectrum of cellular processes  <cit> .
protein kinases modify the targeted protein by transferring phosphate groups from atp or gtp to free hydroxyl groups of serine, threonine or tyrosine in protein amino acid backbone causing conformational change in the protein structure.
it has been estimated that approximately one-third of the eukaryotic proteome is phosphorylated at any given time.
dysregulation of protein kinase-mediated signaling pathways may impair cell growth, proliferation and apoptosis, leading to various disease states  <cit> .
the success of the kinase inhibitor imatinib mesylate  in treatment of selected cancers has generated great interest and hope to use inhibitors of this class of enzymes to treat cancer including promising results with the use of ck <dig> small molecule inhibitors  <cit> .
specificity of phosphorylation by protein kinases is important for the fidelity of signal transduction largely determined by amino acids flanking ser/thr/tyr residues and kinase-substrate concentrations in situ  <cit> .
the constitutively active ck <dig> kinase is the most pleiotropic protein kinase known; it phosphorylates multiple cellular proteins both in vitro and in vivo  <cit> .
ck <dig> is required for cell viability and it is involved in regulation of almost all stages of the cell cycle in yeast and mammals  <cit> .
increased expression of ck <dig> is one of the hallmarks of cancers including the lung, mammary gland, kidney and prostate  <cit> .
this observation has generated great interest and has fueled the search for specific inhibitors of this enzyme.
the atp analog  <dig> -dichloro-1-b-d-ribofuranosylbenzimidazole  was one of the earliest ck <dig> inhibitors used.
modifications of the drb structure by removing the sugar moiety and replacing the chlorines with bromine atoms produced the  <dig> , <dig> -tetrabromo-1h-benzotriazole, tbb.
further reactions within triazole ring generated  <dig> , <dig> -tetrabromo-benzimidazole  and 2-dimethyloamino- <dig> , <dig> -tetrabromo-1h-benzimidazole .
both compounds were shown to be potent ck <dig> inhibitors, in vitro  <cit> .
although ck <dig> inhibitors exhibit different efficacy and specificity, almost all of them inhibit cell proliferation and induce caspase-related apoptosis in the established cancer cell lines  <cit> .
here, we used several assays to examine the mode of action of tbbz and dmat in vivo.
RESULTS
inhibition of cell proliferation by tbbz and dmat in hela cells
the reduction of tetrazolium salts to formazans by living cells results in the color development in the mtt test and reflects the combined effects of cell proliferation and survival.
hela cells were treated with increasing concentrations of tbbz or dmat and mtt test was performed after  <dig> and  <dig> h of the treatment.
the suppressive effect of both ck <dig> inhibitors on cell growth was observed with the highest concentration of inhibitors;  <dig> μm of dmat and  <dig> μm of tbbz .
the results of the mtt test were further confirmed by  thymidine incorporation assays.
again, the proliferation of hela cells was inhibited after  <dig> h  of treatment with  <dig> and  <dig> μm of dmat and tbbz, respectively .
the observed inhibitory effect of both tbbz and dmat on cell proliferation is in agreement with previously published results by pagano et al.
<cit> , however the inhibition efficacy varies considerably between jurkat cells used in that study and the hela cells used here .
tbbz and dmat effects on gene expression profiles
to explore the molecular mechanism of action of these inhibitors in more detail, we performed an oligonucleotide microarray experiment.
we hybridized crna prepared from rna of control hela cells and cells treated for  <dig>   <dig> and  <dig> hours with either  <dig> μm tbbz or  <dig> μm dmat to affymetrix u133a  <dig>  genechip oligo-microarrays containing  <dig> probe sets mapping to  <dig> well-characterized human genes.
in these studies, we established that treatment of serum-starved quiescent cells with 15% of fetal bovine serum changed the level of  <dig> out of  <dig>  transcripts  at least at one of the analyzed time points .
inhibitor treatment for  <dig> and  <dig> hrs mainly caused a decrease in transcript levels , and most of the changes at  <dig> hr of treatment were observed among immediately-early genes .
to confirm microarray data, next we examined the effect of the inhibitors on serum-induced transcription of the selected immediate early genes, egr <dig> , fosb , btg <dig> , nr4a <dig> , nr4a <dig>  and cmyc   <cit> , by rt qpcr.
as shown in figure 2b, serum stimulation of hela cells was accompanied by rapid induction of all transcripts, except cmyc, with a peak increase at 30- <dig> min of stimulation followed by a continual decline to basal expression levels at  <dig> min, the latest experimental time point.
the highest increase was found for egr <dig> mrna , while cmyc transcript increased less than two-fold.
inhibitor treatment significantly decreased the transcript levels at almost all time points.
these results showed an inhibitory effect of both drugs on the induced transcription of immediate early genes suggesting that both agents may inhibit rna polymerase ii  elongation.
this possibility was tested next.
tbbz and dmat inhibit the elongation phase of transcription
transcription complexes of rnapii often pause near the transcription start site   <cit> , and the transition to elongation depends on phosphorylation of the carboxy-terminal domain  of the largest subunit of rnapii.
the ctd consists of multiple heptapeptide repeats with the consensus amino acid sequence  and phosphorylation on ser- <dig> and ser- <dig> residues is mediated by the homologous cyclin dependent kinases , cdk <dig>  - <dig> and - <dig>  erk-1/ <dig>  and c-abl  <cit> .
a potent inhibitor of cdk <dig>  drb behaves as global inhibitor of transcriptionally inducible genes  <cit> .
both tbbz and dmat belong to the class of halogenated imidazole products whose structure was derived from drb.
to establish whether tbbz and dmat arrest elongation phase of transcription, the kinetics of rnapii binding within the egr <dig> gene was further studied using the matrix-chip assay.
rnapii occupancy was assayed at  <dig> positions along the egr <dig> gene, at exon <dig>  and the beginning  and end  of exon <dig>  as well as at a site  <dig> kb downstream of the poly-adenylation  signal .
the latter site was chosen because it was shown for several genes that the rnapii complex continues elongation beyond the poly- site and pauses within  <dig>  -  <dig>  kb downstream.
this process plays an important role in the maturation of the nascent transcript  <cit> .
the chip results show that both tbbz and dmat caused an increase in rnapii occupancy at the first exon of egr <dig> compared to control  but suppressed inducible elongation of rnapii at all regions further downstream after  <dig> min of serum stimulation .
drb exhibited similar inhibitory effects.
this suggested that the inhibitors were preventing rnapii elongation but not recruitment to the egr <dig> promoter.
because both agents inhibited transcription elongation, and both are derivatives of drb, we reasoned that they might affect cdk <dig> kinase activity.
according to the postulated model, transcription initiation of rnapii is regulated by cdk7/cdk8-mediated phosphorylation of ctd at ser- <dig> near the tss.
further down-stream phosphorylation of the ctd at ser- <dig> is required for rnapii to transition to elongation phase  <cit> .
ser <dig> phosphorylation is mediated by cdk <dig>  a kinase that exist in a complex with cyclin t in the positive elongation factor b, p-tefb.
western blot analysis revealed that both tbbz and dmat at a concentration  <dig> μm decreased phosphorylation of the ctd at ser- <dig> residues  while the levels of ctd rnapii were not changed  suggesting inhibition of cdk <dig> kinase activity.
tbbz and dmat inhibit the activity of cdk9
to test the effect of ck <dig> inhibitors on cdk <dig> activity, several in vitro phosphorylation assays were performed.
both tbbz and dmat produced the same dose-dependent decrease in in vitro phosphorylation of hnrnp k protein, a well characterized ck <dig> substrate  <cit>  .
they also inhibited the autophosphorylation of several protein bands when nuclear extracts  were used as a source of both kinase activity and substrate proteins .
densitometric analysis of two highly phosphorylated protein bands revealed that both inhibitors decreased the level of their phosphorylation; tbbz, however, inhibited phosphorylation more efficiently than dmat .
to test the effect of ck <dig> inhibitors on cdk <dig> activity, ne proteins were in vitro phosphorylated with or without inhibitors and then were immunopecipitated with antibodies to both cdk <dig> and its partner cyclin t <dig>  tbbz but not dmat inhibited phosphorylation of precipitated protein with the molecular weight around  <dig> kda, which corresponds to molecular weight of cdk <dig>  these data suggested that tbbz targets components of the p-tefb complex .
the cdk9/cyclin-k complex has also a kinase activity towards the ctd domain of rnap ii and can substitute p-tefb in vitro  <cit> .
thus, we tested the specificity of the drugs inhibitory effect on cdk <dig> autophosphorylation using a recombinant fusion full-length human cdk9-cyclin k protein .
treatment with  <dig> μm tbbz significantly decreased cdk <dig> phosphorylation while the  <dig> μm concentration completely inhibited it .
on the other hand, dmat at concentration of  <dig> μm had little or no effect on cdk <dig> autophosphorylation and at higher concentration  there was a decrease in signal which was less pronounced than that for  <dig> μm tbbz.
to confirm the inhibitory effect of tbbz and dmat on cdk9-mediated phosphorylation of cdk, we carried out additional experiments.
proteins immunoprecipitated  from nuclear extracts  and cdk9-cyclin k recombinant fusion protein complex  were tested in kinase assays using ctd heptapeptide repeats  as a substrate.
both assays revealed that tbbz and dmat inhibited kinase activity of cdk <dig>  although the latter agent was a less effective inhibitor.
discussion
induction of cell proliferation is associated with transcriptional stimulation of growth-related genes that are required for the g1/s transition  <cit> .
the mitogenic stimulation of various cell types is accompanied by rapid induction of immediate-early genes  <cit> .
these genes are involved in cell proliferation, differentiation, apoptosis, and oncogenic transformation  <cit> .
they regulate g1/s transition in the cell cycle and represent diverse functional classes of proteins including transcription factors.
these processes are controlled by external stimuli  <cit>  which activate kinase cascades that transduce mitogenic signals to the nucleus  <cit> .
protein kinases are involved in the regulation of most cellular functions, including cell cycle control, proliferation, and differentiation  <cit> .
ck <dig> is one of the most pleiotropic protein kinases and displays constitutive catalytic activity  <cit> ; it promotes cell survival and plays an anti-apoptotic role  <cit> .
halogenated imidazole class of small molecules are chemotherapeutic candidates but the mode of their action remains poorly understood  <cit> .
in the present study we examined the molecular mechanisms that account for the anti-proliferative activity of two members of this class of drugs, tbbz and dmat.
using microarray analysis we demonstrated that both inhibitors impaired expression of serum induced immediate-early genes, suggesting that these inhibitors block mitogenic signaling cascades.
next, using the matrix-chip assay we found that both compounds inhibited transcription elongation similarly to the known transcription inhibitor, drb, by arresting rnapii near the tss.
although both tbbz and dmat are potent inhibitors, none of the halogenated benzimidazoles affect selectively ck <dig> kinase activity.
in fact, only quinalizarin  which belongs to the same chemical class as emodin, has been shown to be the most potent and selective ck <dig> inhibitor characterized so far, as recently reported by cozza et al.
<cit> .
however, the specificity of ck <dig> inhibitors action in vivo has not been fully characterized.
therefore, our studies may provide a better understanding of the off-target effects of ck <dig> inhibitors.
the routinely used in vitro assays test specificity of ck <dig> inhibitors on a limited set of kinases do not reflect the physiological conditions where other kinases can be directly or indirectly affected by the drugs.
given the pro-survival role of elevated ck <dig> expression in cancer cell lines, a number of studies have been done to determine the effect of ck <dig> inhibitors and their value as anti-cancer agents  <cit> .
a number of cell lines treated with ck <dig> inhibitors responded with the induction of apoptosis through the activation of caspases.
however, the extent of this process and the dose of inhibitor used differed between the cell lines.
with respect to the inhibitors investigated in our study, the viability of hela cells was reduced to 85% after  <dig> hours treatment with dmat, while in another study at the same time point, jurkat cell viability dropped to 25%  <cit> .
contrary to the results with dmat, jurkat cells responded similarly to hela cells when treated with tbbz, with viability reduced to 75% after  <dig> h incubation.
hl- <dig> cells, however, were more potently affected as their viability was decreased to 30% by tbbz treatment  <cit> .
gene transcription in eukaryotes is carried out by the three different dna dependent rna polymerases rnapi, rnapii, and rnapiii.
with regard to the process of transcription, ck <dig> is involved in the regulation of transcription driven by rnapi and rnapiii.
ck <dig> was found at the rdna promoter where it interacts with rnapiβ and phosphorylates several components of the rnapi transcription complex  <cit> .
ck <dig> also plays a fundamental role in the regulation of rnapiii transcription  <cit>  as it binds to the rnapiii complex associated with the u <dig> promoter, and, through phosphorylation of rnapiii associated proteins, plays both positive and negative regulatory roles in the transcription driven by this polymerase  <cit> .
to date, there is less evidence supporting a role of ck <dig> in the regulation of rnapii transcription as compared to the other two polymerases.
it has been shown that phosphorylation of tfiia, tfiie, tfiif  and rnapii by ck <dig> can modify the formation of transcription complexes on the ad-mlp promoter  <cit> .
ck <dig> also phosphorylates fcp <dig>  one of the major phosphatases known to dephosphorylate the rnapii ctd  <cit> .
ck2-dependent phosphorylation greatly enhances the ctd phosphatase activity of fcp <dig> and fcp1-dependent dephosphorylation of the ctd domain is essential for recycling of rnapii and transcription reinitiation  <cit> .
taken together, these observations present ck <dig> as a key factor in the regulation of transcription driven by three nuclear polymerases and we cannot exclude the possibility that the role of ck <dig> in the regulation of rnapii transcription is far greater than currently known.
so, it is possible that inhibitors used here impair immediate-early genes expression, in part, through ck <dig>
the inhibitors used in this study, tbbz and dmat, were previously considered as specific to ck <dig> kinase.
however, according to the results published by pagano et al., both tbbz and dmat appear to be also powerful inhibitors of member of three kinase subfamilies , as well as of pkd <dig>  and cdk <dig>  <cit> .
the spectrum of kinases affected by both inhibitors was similar for both compounds, however the inhibitory effect of dmat on these kinases was slightly higher than that of tbbz.
interestingly, dmat displayed a promiscuous nature inhibiting by > 50%,  <dig> kinases out of  <dig> tested in vitro  <cit> .
this observation is not surprising since the catalytic sites of most human kinases are highly conserved, and cell-permeable kinase inhibitors are mostly competitive with atp for their atp-binding pocket.
thus, inhibitors may similarly target representatives of different branches of the kinome, and the final biological effect of these inhibitors likely reflects functions of the multiple kinases affected.
in eukaryotes, multiple steps of transcription are controlled by phosphorylation of the rnapii ctd domain, mediated by cdks.
cdk <dig> and cdk <dig> regulate the processes involved in transcription initiation while cdk <dig> regulates elongation  <cit> .
recent evidence indicates that a large number of genes are regulated through promoter-proximal pausing  of rnapii.
promoters of some genes with ppp were found to be occupied by the transcriptional machinery but did not produce transcripts, indicating that polymerase recruitment is not rate limiting for expression of those genes.
ppp is particularly prevalent at genes involved in development and in the response to stimuli  <cit> .
the rnapii ctd is hypo-phosphorylated when initially engaged to the promoter, and undergoes serial phosphorylations at ser <dig> during promoter clearance.
this is followed by the binding of drb sensitivity-inducing factor  and negative elongation factor  which, without activity by cdk <dig>  cause rnapii to pause  <cit> .
a classic inhibitor of transcription, drb, which is also the first known inhibitor of ck <dig>  arrests global rnapii-dependent transcription by inhibiting cdk <dig>  which normally phosphorylates the ctd at ser <dig> as well as dsif and nelf  <cit> .
in this case, inhibition of gene transcription by drb may mostly reflect its inhibitory effect on cdk <dig> rather than ck <dig>  in these studies, we demonstrate that tbbz, and at the lesser degree dmat, block phosphorylation of cdk <dig> and its activity in vitro  and both inhibitors decrease ctd phosphorylation in vivo , suggesting the means by which they inhibit rnapii elongation.
knowing the promiscuous nature of the halogenated benzimidazoles it is possible that, apart from cdk <dig>  tbbz and dmat may inhibit also other kinases which activity is essential for rnapii elongation.
CONCLUSIONS
in sum, we used a combination of in vivo and in vitro approaches to evaluate the mode of action of two halogenated imidazole derivative tbbz and dmat.
both agents inhibited cell proliferation and mrna expression and transcription elongation but spectra of their molecular targets may not be the same.
our approach could be used for testing an increasing numbers small molecules derived from dmat/tbb structure, for their affect on transcription elongation by using the matrix-chip assay to asses rnapii density at inducibly transcribed loci.

 phosphorylation is the most common post-translational protein modification that regulates a wide spectrum of cellular processes. protein kinases modify the targeted protein by transferring phosphate groups from atp or gtp to free hydroxyl groups of serine, threonine or tyrosine in protein amino acid backbone causing conformational change in the protein structure. it has been estimated that approximately one-third of the eukaryotic proteome is phosphorylated at any given time. dysregulation of protein kinase-mediated signaling pathways may impair cell growth, proliferation and apoptosis, leading to various disease states. the success of the kinase inhibitor imatinib mesylate in treatment of selected cancers has generated great interest and hope to use inhibitors of this class of enzymes to treat cancer including promising results with the use of ck small molecule inhibitors. specificity of phosphorylation by protein kinases is important for the fidelity of signal transduction largely determined by amino acids flanking ser/thr/tyr residues and kinase-substrate concentrations in situ. the constitutively active ck kinase is the most pleiotropic protein kinase known; it phosphorylates multiple cellular proteins both in vitro and in vivo. ck is required for cell viability and it is involved in regulation of almost all stages of the cell cycle in yeast and mammals. increased expression of ck is one of the hallmarks of cancers including the lung, mammary gland, kidney and prostate. this observation has generated great interest and has fueled the search for specific inhibitors of this enzyme. the atp analog -dichloro-1-b-d-ribofuranosylbenzimidazole was one of the earliest ck inhibitors used. modifications of the drb structure by removing the sugar moiety and replacing the chlorines with bromine atoms produced the, -tetrabromo-1h-benzotriazole, tbb. further reactions within triazole ring generated, -tetrabromo-benzimidazole and 2-dimethyloamino-, -tetrabromo-1h-benzimidazole. both compounds were shown to be potent ck inhibitors, in vitro. although ck inhibitors exhibit different efficacy and specificity, almost all of them inhibit cell proliferation and induce caspase-related apoptosis in the established cancer cell lines. here, we used several assays to examine the mode of action of tbbz and dmat in vivo.  inhibition of cell proliferation by tbbz and dmat in hela cells the reduction of tetrazolium salts to formazans by living cells results in the color development in the mtt test and reflects the combined effects of cell proliferation and survival. hela cells were treated with increasing concentrations of tbbz or dmat and mtt test was performed after and h of the treatment. the suppressive effect of both ck inhibitors on cell growth was observed with the highest concentration of inhibitors; μm of dmat and μm of tbbz. the results of the mtt test were further confirmed by thymidine incorporation assays. again, the proliferation of hela cells was inhibited after h of treatment with and μm of dmat and tbbz, respectively. the observed inhibitory effect of both tbbz and dmat on cell proliferation is in agreement with previously published results by pagano et al., however the inhibition efficacy varies considerably between jurkat cells used in that study and the hela cells used here. tbbz and dmat effects on gene expression profiles to explore the molecular mechanism of action of these inhibitors in more detail, we performed an oligonucleotide microarray experiment. we hybridized crna prepared from rna of control hela cells and cells treated for and hours with either μm tbbz or μm dmat to affymetrix u133a genechip oligo-microarrays containing probe sets mapping to well-characterized human genes. in these studies, we established that treatment of serum-starved quiescent cells with 15% of fetal bovine serum changed the level of out of transcripts at least at one of the analyzed time points. inhibitor treatment for and hrs mainly caused a decrease in transcript levels, and most of the changes at hr of treatment were observed among immediately-early genes. to confirm microarray data, next we examined the effect of the inhibitors on serum-induced transcription of the selected immediate early genes, egr, fosb, btg, nr4a, nr4a and cmyc, by rt qpcr. as shown in figure 2b, serum stimulation of hela cells was accompanied by rapid induction of all transcripts, except cmyc, with a peak increase at 30- min of stimulation followed by a continual decline to basal expression levels at min, the latest experimental time point. the highest increase was found for egr mrna, while cmyc transcript increased less than two-fold. inhibitor treatment significantly decreased the transcript levels at almost all time points. these results showed an inhibitory effect of both drugs on the induced transcription of immediate early genes suggesting that both agents may inhibit rna polymerase ii elongation. this possibility was tested next. tbbz and dmat inhibit the elongation phase of transcription transcription complexes of rnapii often pause near the transcription start site, and the transition to elongation depends on phosphorylation of the carboxy-terminal domain of the largest subunit of rnapii. the ctd consists of multiple heptapeptide repeats with the consensus amino acid sequence and phosphorylation on ser- and ser- residues is mediated by the homologous cyclin dependent kinases, cdk - and - erk-1/ and c-abl. a potent inhibitor of cdk drb behaves as global inhibitor of transcriptionally inducible genes. both tbbz and dmat belong to the class of halogenated imidazole products whose structure was derived from drb. to establish whether tbbz and dmat arrest elongation phase of transcription, the kinetics of rnapii binding within the egr gene was further studied using the matrix-chip assay. rnapii occupancy was assayed at positions along the egr gene, at exon and the beginning and end of exon as well as at a site kb downstream of the poly-adenylation signal. the latter site was chosen because it was shown for several genes that the rnapii complex continues elongation beyond the poly- site and pauses within - kb downstream. this process plays an important role in the maturation of the nascent transcript. the chip results show that both tbbz and dmat caused an increase in rnapii occupancy at the first exon of egr compared to control but suppressed inducible elongation of rnapii at all regions further downstream after min of serum stimulation. drb exhibited similar inhibitory effects. this suggested that the inhibitors were preventing rnapii elongation but not recruitment to the egr promoter. because both agents inhibited transcription elongation, and both are derivatives of drb, we reasoned that they might affect cdk kinase activity. according to the postulated model, transcription initiation of rnapii is regulated by cdk7/cdk8-mediated phosphorylation of ctd at ser- near the tss. further down-stream phosphorylation of the ctd at ser- is required for rnapii to transition to elongation phase. ser phosphorylation is mediated by cdk a kinase that exist in a complex with cyclin t in the positive elongation factor b, p-tefb. western blot analysis revealed that both tbbz and dmat at a concentration μm decreased phosphorylation of the ctd at ser- residues while the levels of ctd rnapii were not changed suggesting inhibition of cdk kinase activity. tbbz and dmat inhibit the activity of cdk9 to test the effect of ck inhibitors on cdk activity, several in vitro phosphorylation assays were performed. both tbbz and dmat produced the same dose-dependent decrease in in vitro phosphorylation of hnrnp k protein, a well characterized ck substrate. they also inhibited the autophosphorylation of several protein bands when nuclear extracts were used as a source of both kinase activity and substrate proteins. densitometric analysis of two highly phosphorylated protein bands revealed that both inhibitors decreased the level of their phosphorylation; tbbz, however, inhibited phosphorylation more efficiently than dmat. to test the effect of ck inhibitors on cdk activity, ne proteins were in vitro phosphorylated with or without inhibitors and then were immunopecipitated with antibodies to both cdk and its partner cyclin t tbbz but not dmat inhibited phosphorylation of precipitated protein with the molecular weight around kda, which corresponds to molecular weight of cdk these data suggested that tbbz targets components of the p-tefb complex. the cdk9/cyclin-k complex has also a kinase activity towards the ctd domain of rnap ii and can substitute p-tefb in vitro. thus, we tested the specificity of the drugs inhibitory effect on cdk autophosphorylation using a recombinant fusion full-length human cdk9-cyclin k protein. treatment with μm tbbz significantly decreased cdk phosphorylation while the μm concentration completely inhibited it. on the other hand, dmat at concentration of μm had little or no effect on cdk autophosphorylation and at higher concentration there was a decrease in signal which was less pronounced than that for μm tbbz. to confirm the inhibitory effect of tbbz and dmat on cdk9-mediated phosphorylation of cdk, we carried out additional experiments. proteins immunoprecipitated from nuclear extracts and cdk9-cyclin k recombinant fusion protein complex were tested in kinase assays using ctd heptapeptide repeats as a substrate. both assays revealed that tbbz and dmat inhibited kinase activity of cdk although the latter agent was a less effective inhibitor. discussion induction of cell proliferation is associated with transcriptional stimulation of growth-related genes that are required for the g1/s transition. the mitogenic stimulation of various cell types is accompanied by rapid induction of immediate-early genes. these genes are involved in cell proliferation, differentiation, apoptosis, and oncogenic transformation. they regulate g1/s transition in the cell cycle and represent diverse functional classes of proteins including transcription factors. these processes are controlled by external stimuli which activate kinase cascades that transduce mitogenic signals to the nucleus. protein kinases are involved in the regulation of most cellular functions, including cell cycle control, proliferation, and differentiation. ck is one of the most pleiotropic protein kinases and displays constitutive catalytic activity; it promotes cell survival and plays an anti-apoptotic role. halogenated imidazole class of small molecules are chemotherapeutic candidates but the mode of their action remains poorly understood. in the present study we examined the molecular mechanisms that account for the anti-proliferative activity of two members of this class of drugs, tbbz and dmat. using microarray analysis we demonstrated that both inhibitors impaired expression of serum induced immediate-early genes, suggesting that these inhibitors block mitogenic signaling cascades. next, using the matrix-chip assay we found that both compounds inhibited transcription elongation similarly to the known transcription inhibitor, drb, by arresting rnapii near the tss. although both tbbz and dmat are potent inhibitors, none of the halogenated benzimidazoles affect selectively ck kinase activity. in fact, only quinalizarin which belongs to the same chemical class as emodin, has been shown to be the most potent and selective ck inhibitor characterized so far, as recently reported by cozza et al.. however, the specificity of ck inhibitors action in vivo has not been fully characterized. therefore, our studies may provide a better understanding of the off-target effects of ck inhibitors. the routinely used in vitro assays test specificity of ck inhibitors on a limited set of kinases do not reflect the physiological conditions where other kinases can be directly or indirectly affected by the drugs. given the pro-survival role of elevated ck expression in cancer cell lines, a number of studies have been done to determine the effect of ck inhibitors and their value as anti-cancer agents. a number of cell lines treated with ck inhibitors responded with the induction of apoptosis through the activation of caspases. however, the extent of this process and the dose of inhibitor used differed between the cell lines. with respect to the inhibitors investigated in our study, the viability of hela cells was reduced to 85% after hours treatment with dmat, while in another study at the same time point, jurkat cell viability dropped to 25%. contrary to the results with dmat, jurkat cells responded similarly to hela cells when treated with tbbz, with viability reduced to 75% after h incubation. hl- cells, however, were more potently affected as their viability was decreased to 30% by tbbz treatment. gene transcription in eukaryotes is carried out by the three different dna dependent rna polymerases rnapi, rnapii, and rnapiii. with regard to the process of transcription, ck is involved in the regulation of transcription driven by rnapi and rnapiii. ck was found at the rdna promoter where it interacts with rnapiβ and phosphorylates several components of the rnapi transcription complex. ck also plays a fundamental role in the regulation of rnapiii transcription as it binds to the rnapiii complex associated with the u promoter, and, through phosphorylation of rnapiii associated proteins, plays both positive and negative regulatory roles in the transcription driven by this polymerase. to date, there is less evidence supporting a role of ck in the regulation of rnapii transcription as compared to the other two polymerases. it has been shown that phosphorylation of tfiia, tfiie, tfiif and rnapii by ck can modify the formation of transcription complexes on the ad-mlp promoter. ck also phosphorylates fcp one of the major phosphatases known to dephosphorylate the rnapii ctd. ck2-dependent phosphorylation greatly enhances the ctd phosphatase activity of fcp and fcp1-dependent dephosphorylation of the ctd domain is essential for recycling of rnapii and transcription reinitiation. taken together, these observations present ck as a key factor in the regulation of transcription driven by three nuclear polymerases and we cannot exclude the possibility that the role of ck in the regulation of rnapii transcription is far greater than currently known. so, it is possible that inhibitors used here impair immediate-early genes expression, in part, through ck the inhibitors used in this study, tbbz and dmat, were previously considered as specific to ck kinase. however, according to the results published by pagano et al., both tbbz and dmat appear to be also powerful inhibitors of member of three kinase subfamilies, as well as of pkd and cdk. the spectrum of kinases affected by both inhibitors was similar for both compounds, however the inhibitory effect of dmat on these kinases was slightly higher than that of tbbz. interestingly, dmat displayed a promiscuous nature inhibiting by > 50%, kinases out of tested in vitro. this observation is not surprising since the catalytic sites of most human kinases are highly conserved, and cell-permeable kinase inhibitors are mostly competitive with atp for their atp-binding pocket. thus, inhibitors may similarly target representatives of different branches of the kinome, and the final biological effect of these inhibitors likely reflects functions of the multiple kinases affected. in eukaryotes, multiple steps of transcription are controlled by phosphorylation of the rnapii ctd domain, mediated by cdks. cdk and cdk regulate the processes involved in transcription initiation while cdk regulates elongation. recent evidence indicates that a large number of genes are regulated through promoter-proximal pausing of rnapii. promoters of some genes with ppp were found to be occupied by the transcriptional machinery but did not produce transcripts, indicating that polymerase recruitment is not rate limiting for expression of those genes. ppp is particularly prevalent at genes involved in development and in the response to stimuli. the rnapii ctd is hypo-phosphorylated when initially engaged to the promoter, and undergoes serial phosphorylations at ser during promoter clearance. this is followed by the binding of drb sensitivity-inducing factor and negative elongation factor which, without activity by cdk cause rnapii to pause. a classic inhibitor of transcription, drb, which is also the first known inhibitor of ck arrests global rnapii-dependent transcription by inhibiting cdk which normally phosphorylates the ctd at ser as well as dsif and nelf. in this case, inhibition of gene transcription by drb may mostly reflect its inhibitory effect on cdk rather than ck in these studies, we demonstrate that tbbz, and at the lesser degree dmat, block phosphorylation of cdk and its activity in vitro and both inhibitors decrease ctd phosphorylation in vivo, suggesting the means by which they inhibit rnapii elongation. knowing the promiscuous nature of the halogenated benzimidazoles it is possible that, apart from cdk tbbz and dmat may inhibit also other kinases which activity is essential for rnapii elongation.  in sum, we used a combination of in vivo and in vitro approaches to evaluate the mode of action of two halogenated imidazole derivative tbbz and dmat. both agents inhibited cell proliferation and mrna expression and transcription elongation but spectra of their molecular targets may not be the same. our approach could be used for testing an increasing numbers small molecules derived from dmat/tbb structure, for their affect on transcription elongation by using the matrix-chip assay to asses rnapii density at inducibly transcribed loci. 
the protein kinase ck plays an important role in diverse biological processes, including cell growth and proliferation as well as in the governing and transduction of prosurvival signals. chip analysis demonstrated that both drugs arrest rnapii elongation. finally, we show that cdk kinase activity, essential for the triggering of rnapii elongation, was blocked by tbbz and to lesser degree by dmat. aberrant activation of protein kinases is one of the essential oncogenic driving forces inherent to the process of tumorigenesis. our approach revealed that small molecules derived from halogenated imidazole compounds may decrease cell proliferation, in part, by inhibiting pathways that regulate transcription elongation. using a high-throughput microarray approach, we demonstrate that two potent inhibitors of ck, -tetrabromo-benzimidazole and 2-dimethyloamino-, -tetrabromo-1h-benzimidazole, blocked mitogen induced mrna expression of immediate early genes.
 aberrant activation of protein kinases is one of the essential oncogenic driving forces inherent to the process of tumorigenesis. the protein kinase ck plays an important role in diverse biological processes, including cell growth and proliferation as well as in the governing and transduction of prosurvival signals. increased expression of ck is a hallmark of some cancers, hence its antiapoptotic properties may be relevant to cancer onset. thus, the designing and synthesis of the ck inhibitors has become an important pursuit in the search for cancer therapies.  using a high-throughput microarray approach, we demonstrate that two potent inhibitors of ck, -tetrabromo-benzimidazole and 2-dimethyloamino-, -tetrabromo-1h-benzimidazole, blocked mitogen induced mrna expression of immediate early genes. given the impact of these inhibitors on the process of transcription, we investigated their effects on rna polymerase ii elongation along the mitogen inducible gene, egr, using chromatin immunoprecipitation assay. chip analysis demonstrated that both drugs arrest rnapii elongation. finally, we show that cdk kinase activity, essential for the triggering of rnapii elongation, was blocked by tbbz and to lesser degree by dmat.  our approach revealed that small molecules derived from halogenated imidazole compounds may decrease cell proliferation, in part, by inhibiting pathways that regulate transcription elongation. 