Activation of the p53-p21(Cip1) pathway is required for CDK2 activation and S-phase entry in primary rat hepatocytes.

p53 plays a major role in the prevention of tumor development. It responds to a range of potentially oncogenic stresses by activating protective mechanisms, most notably cell-cycle arrest and apoptosis. The p53 gene is also induced during normal liver regeneration, and it has been hypothesized that p53 serve as a proliferative 'brake' to control excessive proliferation. However, it has lately been shown that p53 inhibition reduces hepatocyte growth factor-induced DNA synthesis of primary hepatocytes. Here we show that epidermal growth factor (EGF) activated p53 in a phosphatidylinositol-3 kinase-dependent way, and thus induced the cyclin-dependent kinase inhibitor p21(Cip1) in primary rat hepatocytes. p53 inactivation with a dominant-negative mutant (p53(V143A)) attenuated EGF-induced DNA synthesis and was associated with reduced CDK2 phosphorylation and retinoblastoma protein hyperphosphorylation. When p21(Cip1) was ectopically expressed in p53-inactivated cells, these effects were neutralized. In conclusion, our results demonstrate that in normal hepatocytes, EGF-induced expression of p53 is involved in regulating CDK2- and CDK4 activity, through p21(Cip1) expression.

CDK2 regulation through PI3K and CDK4 is necessary for cell cycle progression of primary rat hepatocytes.

INTRODUCTION/OBJECTIVES: Cell cycle progression is driven by the coordinated regulation of cyclin-dependent kinases (CDKs). In response to mitogenic stimuli, CDK4 and CDK2 form complexes with cyclins D and E, respectively, and translocate to the nucleus in the late G(1) phase. It is an on-going discussion whether mammalian cells need both CDK4 and CDK2 kinase activities for induction of S phase. METHODS AND RESULTS: In this study, we have explored the role of CDK4 activity during G(1) progression of primary rat hepatocytes. We found that CDK4 activity was restricted by either inhibiting growth factor induced cyclin D1-induction with the PI3K inhibitor LY294002, or by transient transfection with a dominant negative CDK4 mutant. In both cases, we observed reduced CDK2 nuclear translocation and reduced CDK2-Thr160 phosphorylation. Furthermore, reduced pRb hyperphosphorylation and reduced cellular proliferation were observed. Ectopic expression of cyclin D1 alone was not sufficient to induce CDK4 nuclear translocation, CDK2 activity or cell proliferation. CONCLUSIONS: Thus, epidermal growth factor-induced CDK4 activity was necessary for CDK2 activation and for hepatocyte proliferation. These results also suggest that, in addition to regulating cyclin D1 expression, PI3K is involved in regulation of nuclear shuttling of cyclin-CDK complexes in G(1) phase.

Altered regulation of EGF receptor signaling following a partial hepatectomy.

We have studied epidermal growth factor receptor (EGFR) phosphorylation and localization in the pre-replicative phase of liver regeneration induced by a 70% partial hepatectomy (PH), and how a PH affects EGFR activation and trafficking. When Western blotting was performed on livers after PH with antibodies raised against activated forms of EGFR autophosphorylation sites, no marked increase in EGFR tyrosine phosphorylation was observed. However, events associated with attenuation of EGFR signals were observed. Two hours after PH, we found increased EGFR ubiquitination and internalization, followed by receptor downregulation. Furthermore, EGFR phosphorylation following an injection of EGF was reduced after PH. This reduction correlated with an increased activation of PKC and a distinct augmentation in the phosphorylation of the PKC-regulated T654-site of EGFR. When primary cultured hepatocytes were treated with tetradecanoylphorbol acetate (TPA) to induce T654-phosphorylation of EGFR, we found colocalization of a fraction of EGFR with EEA1, downregulation of EGF-mediated EGFR autophosphorylation, altered ligand-induced intracellular sorting of EGFR, and increased mitogenic signaling through the EGFR-Ras-Raf-ERK pathway. Further, we found that both TPA and a PH enhanced EGF-induced proliferation of hepatocytes. In conclusion, our results suggest that hepatocyte priming involves modulation of EGFR that enhances its ability to mediate growth factor responses without an increase in its receptor tyrosine kinase-activity. This may be a pre-replicative competence event that increases growth factor effects during G1 progression.

Re-localization of activated EGF receptor and its signal transducers to multivesicular compartments downstream of early endosomes in response to EGF.

The rapid internalization of receptor tyrosine kinases after ligand binding has been assumed to be a negative modulation of signal transduction. However, accumulating data indicate that signal transduction from internalized cell surface receptors also occurs from endosomes. We show that a substantial fraction of tyrosine-phosphorylated epidermal growth factor receptor (EGFR) and Shc, Grb2 and Cbl after internalization relocates from early endosomes to compartments which are negative for the early endosomes, recycling vesicle markers EEA1 and transferrin in EGF-stimulated cells. These compartments contained the multivesicular body and late endosome marker CD63, and the late endosome and lysosome marker LAMP-1, and showed a multivesicular morphology. Subcellular fractionation revealed that activated EGFR, adaptor proteins and activated ERK 1 and 2 were located in EEA1-negative and LAMP-1-positive fractions. Co-immunoprecipitations showed EGFR in complex with both Shc, Grb2 and Cbl. Treatment with the weak base chloroquine or inhibitors of lysosomal enzymes after EGF stimulation induced an accumulation of tyrosine-phosphorylated EGFR and Shc in EEA1-negative and CD63-positive vesicles after a 120-min chase period. This was accompanied by a sustained activation of ERK 1 and 2. These results suggest that EGFR signaling is not spatially restricted to the plasma membrane, primary vesicles and early endosomes, but is continuing from late endocytic trafficking organelles maturing from early endosomes.

Cell-specific expression of CCAAT/enhancer-binding protein delta (C/EBP delta) in epithelial lung cells.

CCAAT/enhancer-binding proteins (C/EBP) constitute a family of transcription factors that are involved in regulation of proliferation and differentiation in several cell types. In epithelial lung cells the C/EBP alpha isoform seems to play a role in the regulation of surfactant proteins (SP) and Clara cell specific protein (CCSP), whereas the roles of C/EBP beta and C/EBP delta are unclear. We have examined the protein levels of C/EBP delta in bronchiolar Clara cells and alveolar type 2 cells, and its relation to the expression of lung specific proteins and cell proliferation. The protein expression of C/EBP delta was high in freshly isolated Clara cells compared to type 2 cells. In both cell types C/EBP delta levels increased during culture. Alterations of the levels of C/EBP delta did not correspond with the proliferation levels of Clara cells, but seemed to correspond in type 2 cells. Clara cell secretory protein (CCSP) was highly expressed in freshly isolated Clara cells, in contrast to type 2 cells. SP-D and CYP2B1 were expressed at somewhat higher levels in Clara cells than in type 2 cells, whereas SP-A exhibited highest expression in type 2 cells. During culture the levels of all these lung proteins were strongly reduced. However, compared to with serum we found an increase in CCSP in Clara cell cultures without serum, and this correlated with an increase in C/EBP delta. Overall our in vitro data suggest that C/EBP delta alone is not related to the maintenance of proteins involved in differentiation.

Impaired nuclear accumulation and shortened phosphorylation of ERK after growth factor stimulation in cultured hepatocytes from rats exposed to 2-acetylaminofluorene.

The hepatic carcinogen 2-acetylaminofluorene (AAF) exerts its effect as a tumor promoter by mitoinhibition of normal hepatocytes. Initiated cells proliferate selectively and develop into preneoplastic foci and subsequently into carcinomas. To study whether some of the mitoinhibitory effects of AAF could be attributed to an influence on intracellular signal transduction, growth factor signaling was studied in cultured hepatocytes from rats fed AAF for 7 d. Activation through the epidermal growth factor receptor (EGFR) was used to probe possible changes in downstream mitogenic signaling mechanisms. The proliferative response to epidermal growth factor (EGF), measured as proliferating cell nuclear antigen expression and thymidine incorporation, was almost completely inhibited in hepatocytes exposed to AAF. Neither EGFR protein levels nor EGF binding was notably altered in AAF-exposed hepatocytes as opposed to normal hepatocytes. The initial tyrosine phosphorylation of EGFR and downstream activation of Sos, Raf-1, and extracellular signal-regulated protein kinase (ERK) were similar in AAF-treated and control hepatocytes. Even though ERK phosphorylation was unaffected, a remarkable (80%) reduction of ERK nuclear accumulation was observed in AAF-exposed hepatocytes immediately after mitogen stimulation. EGFR tyrosine phosphorylation and downstream signaling lasted 6 h in control cells versus 2 h in AAF-exposed hepatocytes. We previously demonstrated that AAF inhibits the growth factor-dependent induction of cyclin D1 and arrests hepatocyte cell-cycle progression before the p21/CIP1-controlled DNA-damage check point. The present data indicate that the DNA-damaging carcinogen AAF induces growth inhibition by a distinct inhibition of ERK nuclear accumulation after mitogen stimulation. Inhibition of intracellular signal transduction may represent a novel mechanism of growth arrest. Mol. Carcinog. 28:84-96, 2000.

The carcinogen 2-acetylaminofluorene inhibits activation and nuclear accumulation of cyclin-dependent kinase 2 in growth-induced rat liver.

Growth arrest in G(1) is a common cellular response to DNA damage. In the present study, liver regeneration was combined with continuous exposure for 2-acetylaminofluorene (AAF) to study mechanisms of carcinogen-induced growth arrest in vivo. Growth arrest of uninitiated hepatocytes is central for AAF-induced promotion of premalignant lesions in rat liver. To characterize this growth arrest, we examined the activity of cyclin-dependent kinase (Cdk) 2 in unexposed liver and in AAF-exposed liver after growth induction by partial hepatectomy (PH). Rats were fed either a control diet or an AAF-supplemented diet. After 7 d, a two-third PH was performed and the animals were killed after 0, 12, 18, 24, and 36 h. Kinase assays showed that cyclin E- and Cdk2-associated activities were lower in AAF-exposed liver than in unexposed liver after PH. Although the total cellular levels of cyclin E and Cdk2 were similar, cyclin E-Cdk2 assembly was markedly reduced. In unexposed hepatocytes, Cdk2 translocated to the nuclei after PH. Much of the nuclear Cdk2 was in a rapidly migrating form, presumably representing the Thr160-phosphorylated form of Cdk2. In contrast, in AAF-exposed liver both nuclear Cdk2 accumulation and Thr160-phosphorylation of Cdk2 were reduced. Although p53 and p21(waf1/cip1) were induced by AAF, the binding of p21 to cyclin E and Cdk2 was not increased in growth arrested liver. In conclusion, hepatocyte growth arrest caused by AAF exposure was characterized by a lowered Cdk2 activity that was accompanied by a reduced assembly of cyclin E-Cdk2 complexes but not by binding of p21.

Immunocytochemical localization of Shc and activated EGF receptor in early endosomes after EGF stimulation of HeLa cells.

After binding of epidermal growth factor (EGF), the EGF receptor (EGFR) becomes autophosphorylated via tyrosine. The ligand-activated receptor is internalized by endocytosis and subsequently degraded in the lysosomal pathway. To follow EGFR activation after EGF stimulation, we generated antisera to the EGFR phosphotyrosine sites pY992 and pY1173. The SH2 region of Shc binds to both these sites. Both antisera identified EGFR after EGF binding and did not crossreact with the unactivated receptor. The intracellular distribution of phosphorylated EGFR after ligand binding was traced by two-color immunofluorescence confocal microscopy and immunoelectron microscopy. Before EGF stimulation EGFR was primarily located along the cell surface. When internalization of activated EGFR was inhibited by incubation with EGF on ice, Y992- and Y1173-phosphorylated EGFR were located along the plasma membrane. Ten minutes after internalization at 37C, Y992- and Y1173-phosphorylated EGFR were almost exclusively located in early endosomes, as shown by co-localization with EEA1. Immunoelectron microscopy confirmed that phosphorylated EGFR was located in intracellular vesicles resembling early endosomes. After EGF stimulation, the adaptor protein Shc redistributed to EGFR-containing early endosomes. Our results indicate that EGFR activation of Shc via tyrosine-phosphorylated Y992 and Y1173 occurred in early endocytic compartments, and support a role for membrane trafficking in intracellular signaling.

Alteration of G1 cell-cycle protein expression and induction of p53 but not p21/waf1 by the DNA-modifying carcinogen 2-acetylaminofluorene in growth-stimulated hepatocytes in vitro.

2-Acetylaminofluorene (AAF) is a potent tumor promoter in rat liver carcinogenesis models. In the resistant hepatocyte model, AAF is combined with a growth stimulus for efficient promotion of preneoplastic lesions. The promoting property of AAF in this model is closely associated with mito-inhibition of normal hepatocytes, an effect to which initiated cells are resistant. How AAF induces growth arrest is not known, but genotoxic as well as non-genotoxic effects have been implicated. To elucidate the mechanisms of AAF-induced mito-inhibition, we studied the expression of the tumor suppressor protein p53 and the cyclin-dependent kinase (cdk) complexes mediating G1 progression and S-phase entry. Hepatocytes were isolated from male Fisher 344 rats fed either a control diet or a diet supplemented with 0.02% AAF for 1 wk and cultured in a defined serum-free medium containing epidermal growth factor, insulin, and dexamethasone. Thymidine labeling revealed a profound inhibition of DNA synthesis in AAF-exposed cells compared with control cells. The retinoblastoma protein did not become hyperphosphorylated in AAF-exposed cells. Thus, inhibition of G1 cyclin-cdk activity was implied as a cause of growth arrest. Indeed, G1 cell-cycle arrest was accompanied by reduced induction and nuclear accumulation of the cyclin D1-cdk4 complex and inhibited nuclear translocation of cdk2. Furthermore, the growth arrest was not mediated through p21/waf1 upregulation, although nuclear levels of p53 were increased. Thus, carcinogen-induced mito-inhibition may be effected by altered levels and localization of G1 cyclin-cdk complexes, independent of the upregulation of cdk inhibitory proteins.

[Why is dioxin harmful?]. / Hvorfor er dioksin farlig?

The scandal in Belgium last spring has drawn attention to the environmental hazards of dioxins. Previous production of pesticides and widespread combustion of organic material in the presence of chloride have lead to environmental accumulation of these toxicants, which more precisely are termed polychlorinated dibenzo-p-dioxins and dibenzofurans. Their very long biological half-lives in combination with detectable biological effects at very low concentrations have caused health concerns. Chloracne is the only well documented health effect in man, but there are experimental evidence for carcinogenic, teratogenic, reproductive and immunosuppressive effects. In this presentation we review current knowledge about the cellular effects of dioxins. Dioxins bind to and exert their effects through the cytoplasmic aryl hydrocarbon receptor, which acts as a transcription factor and regulates a number of cytokines and microsomal enzymes. Furthermore, dioxins interfere with hormonal signalling, and anti-oestrogenic effects, vitamin A inhibition and thyroxin mimicry have been reported. Recently, effects on intracellular growth factor signalling have been demonstrated. Dioxins inhibit epidermal growth factor receptor, activate protein kinase C and other intracellular signal transducers, and activate transcription factors. As overall understanding of their cellular mechanisms of toxicity is lacking, we do not possess a complete basis for estimating the adverse health effects of this group of environmental toxicants.

Stress protein expression in rat liver during tumour promotion: induction of heat-shock protein 27 in hepatocytes exposed to 2-acetylaminofluorene.

Exposure of cells to a variety of stresses such as heat, radiation and xenobiotics leads to increased expression of heat-shock proteins (HSPs). HSPs protect cells against irreversible protein damage and are involved in adaptive responses to stress stimuli. Some HSPs are overexpressed in neoplasias, possibly contributing to the increased drug tolerance often observed in such lesions. We have studied HSP expression in two experimental rat hepatocarcinogenesis models. Our aim was to clarify whether they are involved in stress adaptation in hepatocytes during carcinogen exposure, and whether HSPs may contribute to xenobiotic resistance in preneoplastic lesions. The complete carcinogen 2-acetylaminofluorene (AAF) was used in a continuous feeding protocol, and in the resistant hepatocyte model where the growth of diethylnitrosamine initiated lesions is efficiently promoted. Of the HSPs tested, only heat-shock protein 27 (hsp27) was induced during continuous AAF exposure. After 4 weeks of feeding AAF, increased hsp27 expression was noted in hepatocytes in perivenous areas of the liver lobule, possibly mediating an adaptive response to stress caused by reactive AAF metabolites. Enzyme altered preneoplastic foci were not found to overexpress HSPs. Thus, HSP induction does not seem to be a general mechanism underlying the increased stress tolerance observed in such lesions.

Endocytosed epidermal growth factor (EGF) receptors contribute to the EGF-mediated growth arrest in A431 cells by inducing a sustained increase in p21/CIP1.

We investigated the ability of endocytosed activated epidermal growth factor receptors (EGFR) to induce expression of the cyclin-interacting protein p21/CIP1 in A431 cells. Transforming growth factor alpha (TGFalpha) and EGF both induced tyrosine phosphorylation, induction of p21/CIP1, and thereby inhibition of DNA synthesis. TGFalpha is released from the EGFR when the TGFalpha-EGFR complex encounters low pH upon endocytosis. Consistently, we found more rapid dephosphorylation of the EGFR and less induction of p21/CIP1 by TGFalpha than by EGF. This difference was abolished upon neutralizing endosomal pH by the carboxylic ionophore monensin or the proton ATPase inhibitor bafilomycin A1. When surface-bound TGFalpha was removed by acid stripping and endosomal pH was neutralized with bafilomycin A1, TGFalpha stimulated EGFR tyrosine phosphorylation, induced p21/CIP1, and inhibited DNA synthesis. This strongly suggests that p21/CIP1 can be induced by endocytosed, activated EGFR and that endocytosed EGFR can affect cell growth.

Localization of cAMP-dependent signal transducers in early rat liver carcinogenesis.

Cyclic AMP (cAMP) is an important regulator of liver growth and differentiation. The main intracellular cAMP receptor, cAMP-dependent protein kinase (PKA), consists of two regulatory (R) and two catalytic (C) subunits. There are two classes, RI and RII, of the regulatory subunit, giving rise to type I (RI2C2) and type II (RII2C2) PKA. The RI/RII ratio generally decreases during organ development, and increases during carcinogenesis. Alterations in this ratio have been implicated as an important factor in experimental and clinical carcinogenesis. We have studied the expression of RIalpha, RIIalpha, Calpha, and an important substrate of PKA, the cAMP-response element binding protein, during rat liver carcinogenesis. Two-color immunofluorescence and confocal laser scan microscopy were used to characterize localization of the cAMP-dependent signal transducers in hepatocytes, bile ducts, oval cells, and preneoplastic lesions. We found that bile ducts and oval cells (putative liver stem cells) contained a higher RI/RII ratio than hepatocytes and preneoplastic lesions. Thus, an altered RI/RII ratio was not detected during early rat liver carcinogenesis, but may contribute to differentiation of putative liver stem cells to hepatocytes.

Does the mycotoxin citrinin function as a sun protectant in conidia from Penicillium verrucosum.

Our results demonstrate high concentrations of the UV absorbing mycotoxin citrinin in the outer layer of spores from three citrinin-producing strains of Penicillium verrucosum, which is released in an aqueous environment. An important function of the toxin could be to act as a sun protectant in order to create favorable conditions during the initial germination process. When spores from these strains of P. verrucosum were examined by confocal microscopy, a clearly visible fluorescent layer associated with the cell wall was observed. The strains were grown on agar plates, and the mycelial mat was washed with saline. This suspension contained at least 95% of the spores and particulate material, which was removed by filtration after counting the conidia. An aliquot of this filtrate was extracted and citrinin was purified by high pressure liquid chromatography. The absorbance at 319 nm was used to calculate the amount of UV absorbing material released from the spores. Based on the spore numbers in the suspension of the saline extract, we estimated that this material released was 1.4-4.1 pg per spore or 8-24% of the spore weight. Citrinin (and minor amounts of ochratoxin A and some other unidentificable fluorescent compounds) were observed in the filtrate when subjected to thin layer chromatography.

UVB-induced formation of (6-4) photoproducts in the rat corneal epithelium.

The induction of DNA photoproducts in rat corneal epithelium was studied after in vivo exposure to different doses of ultraviolet B light at 297 nm. Affinity-purified antibodies with a major specificity against UV-induced (6-4) photoproducts were used. The results indicate a dose dependent formation of (6-4) photoproducts. Even a minimal erythema dose (25 mJ/cm2) produced (6-4) photoproducts, demonstrating that DNA damage occurs in corneal tissue following exposure to biologically relevant doses of UVB light.

Differential distribution and increased levels of ras proteins during lung development.

Differential localization of ras proteins and variations in their levels may be of importance during lung growth and differentiation. Abundant cell proliferation occurs during development of the fetal rat lung. As assessed by flow cytometry the proliferative activity declined near birth, followed by a gradual increase in cellular proliferation during the subsequent 8 days and a decline to basal levels by 15 to 18 days of age. During this period of substantial variations in proliferative activity, differences in both the protein content and localization of the different ras proteins were observed. The content of N- and K-ras proteins in lung homogenates increased 5 to 6-fold in rats 20 days or older, compared to fetal levels. The protein levels of the ras proteins remained elevated when cellular proliferation decreased to basal levels. As determined by immunohistochemistry, the localization of N-ras protein was restricted to keratin expressing cells of bronchiolar structures, apparently mainly ciliated cells. In contrast, K-ras was found in alveolar cells, probably type I and type 2 cells. H-ras, but not K- or N-ras, was localized to nonepithelial cells. Thus, different ras proteins were localized to different regions of the lung and increased in abundance during postnatal development.

Expression of CYP2B1 in freshly isolated and proliferating cultures of epithelial rat lung cells.

Bronchiolar Clara cells and alveolar type 2 cells of the lung are known to express relatively high levels of P450 enzymes compared to other pulmonary cells. Populations of enriched type 2 cells and Clara cells were isolated from rat lung by a procedure including lung perfusion, protease digestion, centrifugal elutriation, and differential attachment. Alveolar macrophages were removed by lavage. The purity of the type 2 cell-enriched population was approximately 90%, and the purity of the Clara cell-enriched population was 40-50%. Both type 2 cells and the cells of the Clara cell-enriched population proliferated in culture. CYP2B1 mRNA was expressed approximately to the same level in type 2 cells and the Clara cell-enriched population. The mRNA levels remained roughly constant for both cell types throughout the culture period, except for an early transient reduction. The apoenzyme level of CYP2B1 was 2-3 times higher in freshly isolated cells of the Clara cell-enriched population than in the type 2 cells. Both epithelial cell types showed decreased level of CYP2B1 apoenzyme in culture. The differences in the CYP2B1 mRNA and apoenzyme expression levels in freshly isolated cells and cultured cells suggest the existence of a post-transcriptional regulatory mechanism for CYP2B1 expression in lung cells. The characterization of specific functions of lung cells in culture, such as P450 gene expression, provides necessary information for the use of the cells in in vitro pulmonary toxicology.

Growth of colorectal polyps: redetection and evaluation of unresected polyps for a period of three years.

UNLABELLED: BACKGROUND, AIMS, AND PATIENTS: In a prospective follow up and intervention study of colorectal polyps, leaving all polyps less than 10 mm in situ for three years, analysis of redetection rate, growth, and new polyp formation was carried out in 116 patients undergoing annual colonoscopy. The findings in relation to growth and new polyp formation were applied to 58 subjects who received placebo. RESULTS: Redetection rate varied from 75-90% for each year, and was highest in the rectum and sigmoid colon. There was no net change in size of all polyps in the placebo group, however, polyps less than 5 mm showed a tendency to net growth, and polyps 5-9 mm a tendency to net regression in size, both for adenomas and hyperplastic polyps. This pattern was verified by computerised image analysis. Patients between 50 and 60 years showed evidence of adenoma size increase compared with the older patients, and the same was true for those with multiple adenomas (four to five) compared with those with a single adenoma. The new adenomas were significantly smaller and 71% were located in the right side of the colon. Patients with multiple adenomas had more new polyps at all the follow up examinations than patients with a single adenoma. One patient developed an invasive colorectal carcinoma, which may be evolved from a previously overlooked polyp. Two polyps, showing intramucosal carcinoma after follow up for three years, were completely removed, as judged by endoscopy and histological examination. CONCLUSIONS: The results show that follow up of unresected colorectal polyps up to 9 mm is safe. The consistency of growth retardation of medium sized polyps suggests extended intervals between the endoscopic follow up examinations, but the increased number of new polyps in the proximal colon indicates total colonoscopy as the examination of choice. The growth retardation of the medium sized polyps may partly explain the discrepancy between the prevalence of polyps and the incidence of colorectal cancer.

Cyclic AMP-dependent protein kinase (cAK) in human B cells: co-localization of type I cAK (RI alpha 2 C2) with the antigen receptor during anti-immunoglobulin-induced B cell activation.

Cyclic AMP (cAMP) inhibits antigen-stimulated B cell proliferation through activation of cAMP-dependent protein kinases (cAK). We have examined the molecular composition and cellular localization of cAK in human B cells. We find that human B cells contain substantial amounts of mRNA for RI alpha, RII alpha, C alpha and C beta, barely detectable levels of RI beta mRNA, and no detectable RII beta or C gamma mRNA. At the protein level, using Western blotting and subunit-specific antibodies against the different R subunits, we find RI alpha and RII alpha, but no RI beta or RII beta. The presence of catalytic subunits was demonstrated using a nonselective anti-C antiserum. By photoaffinity labeling of R subunits with 8-azido-[32P]cAMP, followed by immunoprecipitation with subunit-specific antibodies, we were also able to demonstrate low levels of RI beta. Immunofluorescence staining of RI alpha and RII alpha demonstrates a rather homogeneous intracellular (but extranuclear) distribution of RI alpha, whereas the RII alpha subunits of cAK are localized to distinct perinuclear structures, previously identified as centrosomes in other cell types. Upon anti-Ig-mediated capping of B cells, RI alpha subunits redistribute to the cap, co-localizing with the antigen-receptors, whereas the intracellular localization of RII alpha subunits remains unchanged.

Differential distribution of Met and epidermal growth factor receptor in normal and carcinogen-treated rat liver.

Transforming growth factor-alpha (TGF-alpha) and hepatocyte growth factor (HGF) are strong hepatocyte mitogens and important regulators of liver regeneration. The TGF-alpha receptor EGFr appears primarily to mediate a proliferative signal, whereas mitogenic, motogenic, and morphogenic effects have been attributed to activation of the HGF receptor Met. We have studied the localization of Met and EGFr in normal and carcinogen-treated rat livers. Oval cells and preneoplastic lesions were induced by diethylnitrosamine initiation, followed by promotion with 2-acetylaminofluorene combined with a partial hepatectomy. Different liver cell populations and their receptor expression were characterized by two-color immunofluorescence and confocal laser scanning microscopy. Hepatocytes were detected by keratin K8 staining, and oval cells and bile ducts were recognized by keratin K19 expression. Enzyme-altered preneoplastic lesions ere identified by expression of placental glutathione S-transferase (GST-pi). Staining for these cellular markers was combined with immunodetection of EGFr and Met. Normal liver exhibited strong staining for EGFr in hepatocytes, whereas blood vessels, bile ducts, and some sinusoidal cells were Met-positive. In carcinogen-treated livers, oval cells showed Met but not EGFr immunostaining. GST-pi-positive foci displayed EGFr immunostaining at a similar intensity as surrounding hepatocytes, whereas Met was not detected. Our data indicate that putative liver cells (oval cells) have a growth receptor phenotype similar to that of bile ducts, whereas preneoplastic live lesions appear hepatocyte-like. These results indicate that the preferential proliferation of preneoplastic liver lesions compared to surrounding hepatocytes is not associated with an altered EGFr or Met phenotype.