Fast electrically assisted regeneration of on-chip SERS substrates.

A microfluidic chip approach utilising integrated electrically connected stationary SERS targets based on inkjet-printed silver nanoparticles is presented. It enables multiple interference-free consecutive surface-enhanced Raman measurements inside chip channels by electrically assisted regeneration of the stationary SERS substrate. Thereby it circumvents common adsorption and memory effect problems associated with stationary SERS targets allowing multiple consecutive measurements in a continuous-flow system.

Contractile tension and beating rates of self-exciting monolayers and 3D-tissue constructs of neonatal rat cardiomyocytes.

The CellDrum technology (The term 'CellDrum technology' includes a couple of slightly different technological setups for measuring lateral mechanical tension in various types of cell monolayers or 3D-tissue constructs) was designed to quantify the contraction rate and mechanical tension of self-exciting cardiac myocytes. Cells were grown either within flexible, circular collagen gels or as monolayer on top of respective 1-mum thin silicone membranes. Membrane and cells were bulged outwards by air pressure. This biaxial strain distribution is rather similar the beating, blood-filled heart. The setup allowed presetting the mechanical residual stress level externally by adjusting the centre deflection, thus, mimicking hypertension in vitro. Tension was measured as oscillating differential pressure change between chamber and environment. A 0.5-mm thick collagen-cardiac myocyte tissue construct induced after 2 days of culturing (initial cell density 2 x 10(4) cells/ml), a mechanical tension of 1.62 +/- 0.17 microN/mm(2). Mechanical load is an important growth regulator in the developing heart, and the orientation and alignment of cardiomyocytes is stress sensitive. Therefore, it was necessary to develop the CellDrum technology with its biaxial stress-strain distribution and defined mechanical boundary conditions. Cells were exposed to strain in two directions, radially and circumferentially, which is similar to biaxial loading in real heart tissues. Thus, from a biomechanical point of view, the system is preferable to previous setups based on uniaxial stretching.

Three-dimensional in vitro reaggregates of embryonic cardiomyocytes: a potential model system for monitoring effects of bioactive agents.

To understand the physiological effects of substances used in drugs and therapies on heart muscle tissue, model systems that mirror the in vivo situation of living tissues are required. Therefore, the creation of 3-dimensional (3D) cell aggregates provides an improved and refined in vitro model as a link between cell-free or single cells and organs or whole organisms in vivo. Here we have characterized a stable contracting in vitro tissue model, which consists of embryonic chicken cardiomyocytes. For establishing a cell-based test system, the 3D in vitro cardiomyocyte spheres were characterized according to messenger RNA expression of special cardiac cell types and protein expression pattern of functional markers such as connexin-43. Finally, the in vitro spheroid model was used for investigating the effect of isoproterenol, a *-adrenergic receptor agonist, on the contractibility mediated by the ligand receptor interaction.

A more aggressive breast cancer spheroid model coupled to an electronic capillary sensor system for a high-content screening of cytotoxic agents in cancer therapy: 3-dimensional in vitro tumor spheroids as a screening model.

One major problem in cancer therapy is the immortality of tumor cells showing an active telomerase, which is responsible for the elongation of the telomeres after each cellular division and the knocking down of apoptotic suppressors. A further phenomenon occurring during cancer therapies is the problem of multicellular resistance. To develop therapeutic anticancer approaches inducing cellular apoptosis, gene-modified biological in vitro systems were established and evaluated for drug screening in a capillary system for a real-time, impedimertic monitoring. Multicellular spheroids of the human breast cancer cell line T-47D clone 11 were transfected with 1) antisense caspase-3 cDNA expression vectors for knocking down the main cell death molecule and 2) sense Bcl-xl cDNA expression vectors for overexpressing the apoptotic suppressor, resulting in more aggressive tumor models. These gene-modified tumor spheroids less sensitive for apoptosis were developed for screening drugs such as methotrexate in tumor spheroid-based biosensor systems via impedance spectroscopy. In this report, it is demonstrated that this could successfully exhibit that this real-time monitoring system with tumor spheroids positioned in a capillary system with a 4-electrode configuration is the most efficient high-content screening module for impedimetric measurements of physiological alterations during gene modification and drug application.

Cytochrome-c oxidase is one of several genes elevated in marginal retina of the chick embryo.

The retinal ciliary margin is particularly relevant for the correct generation and regeneration of vertebrate retinae, since pluripotent stem cells are located there throughout development, and--at least in some species--even until adult stages. Our aim was to identify factors (genes) which are involved in processes of proliferation and differentiation in the developing chicken retina. Reverse transcription-polymerase chain reaction differential display was used to identify genes that were differentially expressed in chick central and peripheral embryonic retina. Candidate genes analyzed through sequencing and database searches were confirmed by Northern blot analysis and histochemistry. A series of differentially expressed genes were detected, including a neuronal cell adhesion molecule, an esterase, and homeobox gene products. One of the sequenced products was identified as subunit I of cytochrome-c oxidase (COX-1), an enzyme which is central to energy metabolism and particularly relevant for developing nervous systems. Northern blot analysis confirmed its up-regulation in the chick peripheral retina, being maximal at embryonic day 7. In the retinal pigmented epithelium its expression is lower than in the retinal periphery but higher than in central retina. COX histochemistry revealed distinct laminar patterns in central retina, but also an elevated level of activity in the peripheral retina throughout development. These data not only show that the developing ciliary margin of the chick retina has high energy requirements, but also indicate that COX-1 could play essential roles in developing cells and in stem cells of the eye periphery.

[Coating material of parlene C as encapsulation material for biomedical micro-implants]. / Untersuchungen zur Abscheidung von Parylen C als Kapselungsmaterial für Biomedizinische Mikroimplantate.

Biomedical microsystems attain to contact with environments like blood, ephitHelium and saline solutions therefore they need an encapsulation. Parylene seems to be a suitable polymer to cover the implants and protect them against moisture and aggressive environment. This paper describes the characterisation of Parylene C coatings and its possibilities to protect implants. Beside the encapsulation of biomedical microsystems the manufacturing of fexible electrodes and the cytotoxicity behavior of RIE etched Parylene layers was investigated.

Heterologous expression of acetylcholinesterase affects proliferation and glial cytoskeleton of adherent chicken retinal cells.

Besides its function at cholinergic synapses, acetylcholinesterase (AChE) exerts structural functions on neural differentiation, independent of its enzymatic activity. To elucidate such functions, we have previously heterologously expressed AChE in histotypic retinal reaggregates, revealing strong effects on their histogenesis, particularly on Müller glia processes. To further resolve these findings at a less complex cellular level, in this study we transfected adherent retinal cells of the chick embryo after 2 days i.c. with a sense pSVK3-AChErab-cDNA expression vector encoding for the entire rabbit AChE gene by calcium phosphate precipitation. Northern blots using digoxigenin (DIG)-labeled rabbit cDNA revealed a pronounced level of rabbit AChE mRNA in AChE-transfected cells. Western blot analysis established an increase in the endogenous AChE protein in transfected cells. Noticeably, AChE activity was not much affected, indicating a post-translational regulation of overall AChE activity. As a corollary, 5'-bromo-2'-deoxyuridine (BrdU) studies showed a decrease in cell proliferation. Exploring changes of the Müller glia, the cytoskeletal protein vimentin was found to be increased in transfected cells. Vimentin-stained processes are longer, thicker and more orderly arranged. In conclusion, exogenous expression of rabbit AChE in chicken retinal monolayers exerts a structural function on glial cytoskeletal organization, independent of AChE activity.

Anticholinesterase treatment of chicken retinal cells increases acetylcholinesterase protein independently of protein kinase C.

It has been reported that anticholinesterase exposure, e.g. by environmental toxins or nerve gases, can increase acetylcholinesterase (AChE) protein, possibly as an autoregulatory stress response. We earlier have transfected retinal cells of the chick embryo with a pSVK3-AChE(rab)-cDNA vector to heterologously express rabbit AChE, which concomitantly also increased AChE protein from chick. To analyse further the cell-internal pathways of these different paradigms (anticholinesterase treatment vs. AChE transfection) which both lead to an AChE increase, we here show that AChE overexpression by transfection leads to an increase in protein kinase C (PKC). Most remarkably, when cells independently of, or in addition to their transfection are treated with 10 microM of the AChE inhibitor BW284c51, AChE protein levels are much more dramatically increased up to 20-fold. This treatment, however, does not affect PKC. These data show that (i) retinal cells respond to anticholinesterase insult by a massive increase of AChE protein; (ii) the response to BW284c51 is not PKC-mediated; and (iii) both strategies of AChE increase follow different cell-internal pathways, their effects being additive. The ecological and biomedical implications of these findings are briefly discussed.

A multicellular spheroid-based sensor for anti-cancer therapeutics.

The progress in cellular engineering offers novel approaches for anti-cancer therapies. To investigate the effectiveness of potential therapies efficient screening methods are required. We propose an impedance measurement system which enables the use of multicellular spheroid models in bioelectronic screening systems either for non destructive life-time diagnostic or anti-cancer therapies. A biohybrid sensor system is created comprising gene-manipulated T47D clone 11 breast carcinoma spheroids positioned hydrodynamically in a capillary system with electrodes. A novel approach employing an antisense-5'butyrylcholinesterase expression system is probed on reaggregated tumor cells under simulated microgravity, inhibiting the gene transcription and translation of the embryonic proliferation marker butyrylcholinesterase expressed in different tumor types. Alterations in the morphology of cell aggregates e.g. apoptosis or necrosis can be detected by impedance spectroscopy monitoring the electric behavior of membranes and extracellular space with a high resolution and reproducibility. The hydrodynamic positioning of 3D in vitro cell aggregates and the short time for the measurements represent an innovative method for a synchronized multicapillary screening system. The combination of the measuring system with a bioreactor enables cyclic life time recordings of impedance spectra for monitoring the cell aggregate properties for a long period.

Biohybrid microarrays--impedimetric biosensors with 3D in vitro tissues for toxicological and biomedical screening.

To investigate the effectiveness of potential anticancer therapeutics or therapies, efficient screening methods are required. On the one hand, multicellular 3D aggregates (spheroids) are a powerful in vitro model for simulating the in vivo situation and on the other hand, planar electrode structures are generally highly suitable for automation and parallel testing. Here, the detection of the effect of active substances on spheroids positioned on electrodes of substrate integrated electrode arrays is exemplarily investigated. As a 3D tissue model a reaggregation system of T47D clone 11 tumor cells is used. The effect of cytotoxins (DMSO, Triton X-100) on spheroids can be detected by recording the effective impedance of planar electrodes covered by spheroids. The equivalent circuit model parameter of electrodes covered by cytotoxin treated spheroids are determined from recorded impedance spectra and compared to the parameter of electrodes covered by control spheroids as well as not covered electrodes. Spheroids on electrodes mainly influence the electrode impedance in the frequency range of 10 kHz to 1 MHz. The results are discussed in view of an optimal electrode/spheroid-interface for sensing the effects of therapeutics with high sensitivity.

Regulation of the rat oligodendroglia cell line OLN-93 by antisense transfection of butyrylcholinesterase.

Butyrylcholinesterase (BChE) is a glial cell marker with unknown function. For neuroepithelial cells, BChE has been shown to regulate cell division and expression of the postmitotic marker acetylcholinesterase (AChE), while similar studies are lacking for glial cells. By transducing an antisense-5'BChE cDNA expression vector via calcium phosphate precipitation, we have analyzed the effect of BChE inhibition on proliferation and differentiation of rat oligodendroglia-derived OLN-93 cells. OLN-93 cells were chosen because they are highly proliferative, while expressing markers of differentiated oligodendrocytes (Richter-Landsberg and Heinrich, 1996). First, we established that OLN-93 cells do express BChE protein, albeit chiefly in an inactive state, and that BChE was decreased by antisense-5'BChE transfection. Cell proliferation was also strongly diminished, protein kinase C (PKCalpha) was upregulated, and expression of cytoskeletal and cell surface proteins was altered. In particular, immunoreactivities of the intermediate filament proteins vimentin and the cell adhesion protein F11 were detected, indicating that BChE-inhibited OLN-93 cells have shifted toward an astrocytic phenotype. These data support a role of the glia marker BChE in CNS glial cell proliferation and differentiation, achieved via a nonenzymatic mechanism. The possible biomedical impact of BChE protein, e.g., on CNS nerve regeneration, is briefly discussed.

The key role of butyrylcholinesterase during neurogenesis and neural disorders: an antisense-5'butyrylcholinesterase-DNA study.

The wide tissue distribution of butyrylcholinesterase (BChE) in organisms makes specific roles possible, although no clear physiologic function has yet been assigned to this enzyme. In vertebrates, it appears e.g. in serum, hemopoietic cells, liver, lung, heart, at cholinergic synapses, in the central nervous system. in tumors and not at least (besides acetylcholinesterase, AChE) in developing embryonic tissues. Here, a functional role of BChE can be found in regulation of cell proliferation and the onset of differentiation during early neuronal development--independent of its enzymatic activity. For studies concerning this point, we have established a strategy for a specific and efficient inhibition of BChE to investigate how the expected decrease of enzyme and, therefore, the manipulation of cellular cholinesterase-equilibrium influences embryonic neurogenesis--among others to gain information about the significance of noncholinergic, activity-independent and cell growth functions of BChE. The antisense-5'BChE-DNA strategy is based on inhibition of BChE mRNA transcription and protein synthesis. For this, the BChE gene is cloned into a suitable vector system; this is done in antisense-orientation, so that a transfected cell will produce their own antisense mRNA to inhibit gene expression. For such investigations in neurogenesis, the developing retina is a good model and we are able to create organotypic, three-dimensional retinal aggregates in vitro (retinospheroids) using isolated retinal cells of 6-day-old chicken embryos. Using this in vitro retina and "knock out" of BChE gene expression, we could show a key role of BChE during neurogenesis. The results are of great interest because in tumorigenesis and some neuronal disorders, the BChE gene is amplified or abnormally expressed. It has to be discussed how the antisense-5'BChE strategy can play a role in the development of new and efficient therapy forms.

Fucose in alpha(1-6)-linkage regulates proliferation and histogenesis in reaggregated retinal spheroids of the chick embryo.

We have used the lectin from Aleuria aurantia (AAL) which is highly specific for alpha(1-6)-linked fucose, to examine its effect on chicken retinogenesis in a reaggregation culture system. When dispersed cells of the embryonic chick retina are reaggregated to form histotypic retinospheroids, AAL elicits strong inhibition of spheroid growth. The action of AAL is specific, since its effect is dose-dependent, saturable, and inhibited by an excess of fucose. Fucosidase treatment entirely abolishes reaggregation. In contrast, Anguilla anguilla agglutinin (AAA) binding to fucose in alpha(1-2)-linkage does not show any effects. Incubation with CAB4-a specific monoclonal antibody for fucose in alpha(1-6)-linkage-reduces spheroid size and shape. AAL does not much affect primary aggregation, but rather subsequent processes of cell proliferation and histogenesis. In particular, AAL inhibits uptake of bromo-desoxyuridine (BrdU), most efficiently so during days in vitro 2 (div2) and div3. As a consequence, the histological differentiation is entirely disturbed, as evidenced by vimentin immunostaining; particularly, rosettes are not forming and the radial glia scaffold is disorganized. We conclude that glycoproteins exhibiting fucose in alpha(1-6)-linkage may play major roles in early processes of retinal tissue formation.

Butyrylcholinesterase is complexed with transferrin in chicken serum.

The function of the enzyme butyrylcholinesterase (BChE) both in serum and in brain is unclear. In serum, BChE has been found complexed with several biomedically relevant proteins, with which it could function in concert. Here, the existence of a similar complex formed between BChE and sero-transferrin from adult chicken serum was elucidated. In order to identify both proteins unequivocally, we improved methods to highly purify the 81-kDa BChE and the coisolated 75-kDa transferrin, which then allowed us to tryptically digest and sequence the resulting peptides. The sequences as revealed for BChE peptides were highly identical to mammalian BChEs. A tight complex formation between the two proteins could be established (a) since transferrin is coisolated along with BChE over three steps including procainamide affinity chromatography, while transferrin alone is not bound to this affinity column, and (b) since imunoprecipitation experiments of whole serum with a transferrin-specific antiserum allows us to detect BChE in the precipitate with the BChE-specific monoclonal antibody 7D11. The possible biomedical implications of a complex between transferrin and BChE which here has been shown to exist in chicken serum are briefly discussed.

Butyrylcholinesterase antisense transfection increases apoptosis in differentiating retinal reaggregates of the chick embryo.

To investigate the roles of the enzymes butyryl- and acetylcholinesterase (BChE and AChE) in retinal proliferation and differentiation, we use reaggregated spheres from retinal cells of the 6-day-old chick embryo, forming cellular and fibrous areas homologous to all layers of a normal retina. Recently, we could suppress BChE expression by transfecting these so-called retinospheroids during their proliferation period with a pSVK3 expression vector containing a 5' fragment of the rabbit BChE gene in antisense orientation. Along with morphological changes, proliferation was significantly decreased. Here, we have studied the effect of antisense BChE suppression during the differentiation period of retinospheroids. As BChE is suppressed, the differentiation of AChE-positive cells is increased, whereas the immunoreactivities for red and green cone-specific opsins are strongly reduced. Concomitantly, the rate of apoptosis as determined by propidium iodide uptake, by increased CPP 32-like caspase expression, and by terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling and DNA fragmentation assays is roughly doubled, predominantly at the expense of degenerating photoreceptor precursors. This is further strong evidence that the proliferation marker BChE regulates an intricate balance between cell proliferation, cell differentiation, and programmed cell death in this in vitro retinal system.

Aryl acylamidase activity exhibited by butyrylcholinesterase is higher in chick than in horse, but much lower than in fetal calf serum.

Several side activities have been attributed to butyrylcholinesterase (BChE), including aryl acylamidase (AAA) activity, which is an amidase-like activity with unknown physiological function splitting the artificial substrate o-nitroacetanilide. For avians, extensive developmental data have pointed to neurogenetic functions of BChE, however, a possible AAA activity of BChE has not been studied. In this study, we first compare the relative levels of AAA exhibited by BChE in whole sera from chick, fetal calves (FCS) and horse. Remarkably, FCS exhibits a 400-fold higher ratio of AAA/BChE than horse and 80-fold higher than chick serum. We then show that an immunoisolated preparation of BChE from chicken serum presents significant activity for AAA. Both in sera and with the purified enzyme, the AAA activity is fully inhibited by anticholinesterase drugs, showing that AAA activity is exclusively conveyed by the BChE molecule. Noticeably, AAA inhibition even occurs at lower drug concentrations than that of BChE activity itself. Moreover, AAA is sensitive to serotonin. These data indicate that (1) AAA is a general feature of serum BChE of vertebrates including avians, (2) AAA is effectively inhibited by cholinergic and serotonergic agents, and (3) AAA may have a developmental role, since it is much pronounced in a serum from fetal animals. Functionally, deamination of neuropeptides, a link between cholinergic and serotonergic neurotransmitter systems, and roles in lipoprotein metabolism could be relevant.

Transfection of reaggregating embryonic chicken retinal cells with an antisense 5'-DNA butyrylcholinesterase expression vector inhibits proliferation and alters morphogenesis.

The function of the enzyme butyrylcholinesterase (BChE) in the developing and mature brain is still unclear. We have inserted 577 bp of the 5' upstream region plus 106 bp of the exon 1 of the rabbit BChE gene in reverse orientation under control of an SV40 early promoter derivative in an expression vector. This vector was introduced by calcium phosphate-mediated transfection into embryonic chicken retina cells during the first days of reaggregation culture. Depending on the retinal origin, the transfected cell population forms histotypic retina-like spheres, so-called rosetted or stratified retinospheroids. We show that antisense 5'-BChE gene expression decreased the steady-state mRNA level of BChE and the translation of the BChE protein, inhibited proliferation, and accelerated histogenesis in both cellular systems. The pronounced effects of antisense 5'-BChE transfection of spheroids document a key role of BChE during the early reaggregation process of retinal cells, most likely by regulating their growth and differentiation.

Regulation of cholinesterase gene expression affects neuronal differentiation as revealed by transfection studies on reaggregating embryonic chicken retinal cells.

In the embryonic chicken neuroepithelium, butyrylcholinesterase (BChE) as a proliferation marker and then acetylcholinesterase (AChE) as a differentiation marker are expressed in a mutually exclusive manner. These and other data indicate a coregulation of cholinesterase expression, and also possible roles of cholinesterases during neurogenesis. Here, both aspects are investigated by two independent transfection protocols of dissociated retina cells of the 6-day-old chick embryo in reaggregation culture, both protocols leading to efficient overexpression of AChE protein. The effect of the overexpressed AChE protein on the re-establishment of retina-like three-dimensional networks (so-called retinospheroids) was studied. In a first approach, we transfected retinospheroids with a pSVK3 expression vector into which a cDNA construct encoding the entire rabbit AChE gene had been inserted in sense orientation. As detected at the mRNA level, rabbit AChE was heterologously overexpressed in chicken retinospheroids. Remarkably, this was accompanied by a strong increase in endogenous chicken AChE protein, while the total AChE activity was only slightly increased. This increase was due to chicken enzyme, as shown by species-specific inhibition studies using fasciculin. Clearly, total AChE activity is regulated post-translationally. As an alternative method of AChE overexpression, transfection of spheroids was performed with an antisense-5'-BChE vector, which not only resulted in the down-regulation of BChE expression, but also strongly increased chicken AChE transcripts, protein and enzyme activity. Histologically, a higher concentration of AChE protein (as a consequence of either AChE overexpression or BChE suppression) was associated with an advanced degree of tissue differentiation, as detected by immunostaining for the cytoskeletal protein vimentin.

Recombinant human casein kinase II. A study with the complete set of subunits (alpha, alpha' and beta), site-directed autophosphorylation mutants and a bicistronically expressed holoenzyme.

Human casein kinase II (CKII) is a ubiquitous and multipotential Ser/Thr kinase involved in the regulation of cell growth and differentiation. Biochemically, two characteristics are particularly notable; first, the tetrameric composition of two catalytic subunits (alpha and/or alpha') and two regulatory subunits (beta); second, the autophosphorylation of the holoenzyme at the N-terminus of CKII beta, suspected to be involved in tuning of the kinase activity. Whether CKII alpha and CKII alpha' reconstitute comparably with CKII beta to form holoenzyme is unclear. For a systematic investigation, the complete set of recombinant CKII subunits and of autophosphorylation mutants of CKII beta were expressed in Escherichia coli and comparative reconstitutions carried out. At 1:1 molar ratio, CKII beta stimulated both catalytic subunits roughly fivefold with phosvitin as a substrate. The level of activity reached with both of the reconstituted CKII isoforms was of the same order of magnitude as that of holoenzyme isolated from human placenta. It was also similar to a recombinant alpha 2 beta 2 holoenzyme whose expression had been attained in E. coli with a bicistronic construct containing the coding regions of CKII beta and CKII alpha in a tandem arrangement. Both Ser2 and Ser3 were identified as the autophosphorylation sites; replacement of one of these with Ala by oligonucleotide-mediated site-directed mutagenesis influenced only the extent of CKII beta autophosphorylation, replacement of both resulted in a loss of autophosphorylation. Despite these differences, the stimulatory effect of all the CKII beta mutants was comparable both to each other and to that of wild-type CKII beta. This was also obtained when substrates other than phosvitin were employed such as tubulin, or upstream-binding factor (UBF). However, the degree of stimulation was substrate specific and ranged from 2-5-fold with no major differences between CKII alpha and CKII alpha' stimulation. Calmodulin phosphorylation by both CKII alpha and CKII alpha' was decreased similarly by CKII beta and the CKII beta mutants. Proteins such as cAMP-responsive-element-binding protein (CREB), HPV16 E7 or Jun were not phosphorylated by either catalytic subunit but became substrates of both in the presence of CKII beta or CKII beta mutants. The data suggest that CKII alpha and CKII alpha' form similar CKII holoenzymes and that the tuning of holoenzyme activity is independent of the autophosphorylation status of CKII beta.

Human casein kinase II. The subunit alpha protein activates transcription of the subunit beta gene.

Casein kinase II (CKII), an ubiquitous serine/threonine protein kinase in control of a variety of crucial cellular functions, is composed of catalytic subunits (alpha and alpha') and regulatory subunits (beta). The adjusted activity of CKII is determined by the actual conformational state of CKII beta and the stoichiometry of the CKII subunits. Thus, the expression control of CKII beta is of particular concern. Carrying out gel shifts and footprints with affinity-purified proteins and cellular extracts in combination with mutational analysis we find that aside NF1 and Sp1, two out of the many factors predicted to bind to the upstream promoter region of the human CKII beta gene (Voss, H., Wirkner, U., Jakobi, R., Hewitt, N. A., Schwager, C., Zimmermann, J., Ansorge, W., and Pyerin, W. (1991) J. Biol. Chem. 266, 13706-13711), CKII alpha protein is able to complex with the CKII beta gene promoter. The complex of CKII beta-DNA/CKII alpha-protein is shown to occur within the 170-239-base pair (bp) segment upstream of the first transcription start site of the gene. The DNA motif contains, in a distance of 44 bp, two GC-rich boxes, 5'-GGGGCCC and 5'-CCCCTGGGC, and represents a novel cis-acting element; the binding of the CKII alpha protein activates the CKII beta gene promoter. This is manifested by driving the expression of the indicator gene luciferase or of CKII alpha-cDNA in HeLa cells. The binding of the CKII alpha protein is inhibited due to CKII beta protein addition or by mimicking the corresponding situation in vivo by overexpression of the CKII subunits. The data suggest that cells may maintain a certain CKII subunit stoichiometry via transcriptional control; excess of nuclear CKII alpha protein could activate the CKII beta gene transcription causing CKII beta protein to increase which, in turn, could feed back to abolish the action of CKII alpha at the CKII beta gene promoter.