Synergistic effects of endothelin-1 (ET-1) and transforming growth factor alpha (TGF-α) or epidermal growth factor (EGF) on DNA replication and G1 to S phase transition

The cooperative cell kinetic actions of ET-1 with TGF- or EGF in normal rat kidney fibroblasts (NRK-49F) and KNRK cells (Kirsten MSV transformed) were analyzed by [³H]-thymidine incorporation assay and flow cytometry. A marked synergistic effect of TGF- and ET-1 (or EGF and ET-1) on DNA synthesis and G1 to S transition was observed in NRK cells; 15–20% S for TGF- and 12% S for ET-1 alone but 45–50% S in combination. There was no detectable effect on cell cycle kinetics by TGF- (1 ng/ml) or EGF (1 ng/ml) plus ET-1 (1 ng/ml) in KNRK cells treated for 22 hours. Insulin, insulin-like growth factor I (IGF-I), fibroblast growth factor (FGF), platelet derived growth factor (PDGF), and transforming growth factor (TGF-) were also tested and found to have no significant synergistic effects on ET-1 actions. Our findings suggest that the combination of TGF- (EGF) and ET-1 is an important part of an intricate network which coordinates progression of G1 to S phase in normal cells.     

The Expression of Transforming Growth Factor Alpha Receptor Protein and its Activation in Chicken Ovarian Granulosa Cells of Maturing Follicles

Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) are structurally related growth factors that exert their biological actions by binding to the same cell-surface receptor, EGF receptor. However, in chicken cells, human EGF binds with approximately 100-fold lower affinity than human TGF-. In a previous study, we localized EGF/TGF- receptor immunohistochemically in the granulosa and theca of the developing follicles of laying hens. We have also shown that TGF- binds to cell-surface receptors of the granulosa cells. The present study characterizes the nature of the EGF/TGF- receptor. Immunoprecipitation of receptor proteins from cultured granulosa cells with an anti-EGF receptor antibody (12E) shows the expression of a 170-kDa receptor protein. The expression of the receptor protein decreases with follicular enlargement between the F3 and F1. Incubation of the cells with [125I]TGF- followed by crosslinking with bis(sulphosuccinimidyl)suberate showed that TGF- binds a similar (170 kDa) receptor protein immunoprecipitated with the 12E anti-EGF receptor antibody. The binding of TGF- to granulosa cells caused receptor protein oligomerization, yielding the monomeric (170 kDa) and dimeric (340 kDa) protein forms. Oligomerization seemed to favour the formation of the dimeric rather than the monomeric form. Culturing granulosa cells with luteinizing hormone or follicle-stimulating hormone increased the expression of both monomer and dimer forms of the receptor proteins compared with the control. Western blotting analysis with anti-phosphotyrosine antibody revealed that the lysates of TGF--stimulated cells express phosphotyrosine-containing receptor proteins of 170 kDa and 340 kDa. The results show that chicken granulosa cells express the 170-kDa EGF=TGF- receptor protein, which dimerizes on binding to TGF-, suggesting that the receptor protein may be involved in the signal transduction of TGF- actions in the chicken granulosa cells.     

In vitro secretion of transforming growth factor alpha (TGF-α): A comparison of the A431 cell line with three human oesophageal squamous cell carcinoma lines

Transforming growth factor alpha (TGF-) is a single chain polypeptide which exists in a variety of forms differing in molecular weight. These forms are variously present in normal and neoplastic cells. Of particular interest are TGF-'s well-known mitogenic properties. The transition from a normal to a neoplastic cellular state results from signalling defects that may depend upon,iter alia, abonormal levels of expression and secretion of TGF-. It is known that the secretion of TGF- may be enhanced appreciably by agents such as phorbol 12-myristate 13-acetate (PMA), serum factors and epidermal growth factor (EGF). Here, we compare the efficacy of these three agents in the elevation of TGF- secretion in the well studied A431 cell line with their previously undocumented efficacy in certain interesting, but little known, human oesophageal squamous cell carcinoma (SCC) lines.     

Role of protein kinase C in the regulation of cytosolic Ca2+ in A431 cells: Separation of growth factor and bradykinin pathways

Summary Calcium signaling systems in nonexcitable cells involve activation of Ca²⁺ entry across the plasma membrane and release from intracellular stores as well as activation of Ca²⁺ pumps and inhibition of passive Ca²⁺ pathways to ensure exact regulation of free cytosolic Ca²⁺ concentration ([Ca²⁺] _i ). A431 cells loaded with fura-2 cells were used as a model system to examine regulation of Ca²⁺ entry and intracellular release. Epidermal growth factor (EGF) and transforming growth factor alpha (TGF-) both stimulated Ca²⁺ entry and release while bradykinin appeared only to release Ca²⁺ from intracellular stores. The possible role of protein kinase C (PKC) in modulating the [Ca²⁺] _i response to these agonists was examined by four methods. Low concentrations of TPA (2×10^–10 m) had no effect on Ca²⁺ release due to EGF, TGR- or bradykinin but resulted in a rapid return of [Ca²⁺] _i to baseline levels for EGF or TGF-. Addition of the PKC inhibitor staurosporine (1 and 10nm)_completely inhibited the action of TPA on EGF-induced [Ca²⁺] _i changes. An inhibitor of diglyceride kinase (R59022) mimicked the action of TPA. Down-regulation of PKC by overnight incubation with 0.1 or 1 m TPA produced the converse effect, namely prolonged Ca²⁺ entry following stimulation with EGF or TGF-. To show that one effect of TPA was on Ca²⁺ entry, fura-2 loaded cells were suspended in Mn²⁺ rather than Ca²⁺ buffers. Addition of EGF or TGF- resulted in Ca²⁺ release and Mn²⁺ entry. TPA but not the inactive phorbol ester, 4--phorbol-12,13-didecanoate, inhibited the Mn²⁺ influx. Thus, PKC is able to regulate Ca²⁺ entry due to EGF or TGF- in this cell type. A431 cells treated with higher concentrations of TPA (5×10^–8 m) inhibited not only Ca²⁺ entry but also Ca²⁺ release due to EGF/TGF- but had no effect on bradykinin-mediated Ca²⁺ release, suggesting differences in the regulation of the intracellular stores responsive to these two classes of agonists. Furthermore, sequential addition of EGF or TGF- gave a single transient of [Ca²⁺] _i , showing a common pool of Ca²⁺ for these agonists. In contrast, sequential addition of EGF (or TGF-) and bradykinin resulted in two [Ca²⁺] _i transients equal in size to those obtained with a single agonist. Ionomycin alone was able to fully deplete intracellular Ca²⁺ stores, whereas ionomycin following either EGF (or TGF-) or bradykinin gave an elevation of the [Ca²⁺] _i signal equal to that of the second agonist. These data indicate that there are separate pools of intracellular Ca²⁺ for EGF-mediated Ca²⁺ release which also respond differently to TPA.     

Interleukin-6 Enhances Transforming Growth Factor-alpha mRNA Expression in Macrophage-Like Human Monocytoid (U-937-1) Cells

We have previously reported that the human monocytoid cell line U-937-1 constitutively expresses transforming growth factor-alpha (TGF-) and that the steady-state levels of TGF- mRNA as well as TGF- protein release increase when U-937-1 cells are differentiated towards monocytes/macrophages. Interleukin-6 (IL-6), which has been shown to have growth-stimulatory effects on a number of cell types, has recently been shown to enhance TGF- expression in keratinocytes. In the present study we investigated whether TGF- expression in macrophage-like cells could be regulated by IL-6 using U-937-1 cells as a model system of monocyte/macrophage differentiation.U-937-1 cells were differentiated with retinoic acid (RA), vitamin D3 (Vit-D3) or phorbol-12-myristate-13-acetate (PMA) for 4 days and were then treated with human recombinant IL-6 (1000 IU/ml) for up to 24 hr. Northern blot analysis revealed that cells differentiated with PMA, inducing the phenotype of a secretory macrophage, markedly increased their TGF- mRNA levels (2.7-fold) when treated with IL-6; the response was maximal at 6 hr and remained high at 12 hr. The expression of the TGF- gene was accompanied by release of TGF- protein into the cell culture medium, irrespective of differentiating agent, as demonstrated by enzyme-linked immunosorbent assay (ELISA), as well as by surface expression of pro-TGF- as determined by indirect immunofluorescent cytometry. However, the superinduction of the TGF- gene by IL-6 in cells differentiated with PMA was not accompanied by any increase in TGF- protein release or pro-TGF- surface expression.We conclude that since IL-6 causes increased steady-state levels of TGF- mRNA in macrophage-like cells, it may prime these cells for production of this growth factor. Furthermore, we have shown that the IL-6 receptor complex is functional in U-937-1 cells induced to differentiate towards a secretory macrophage by treatment with PMA.     

Developmental expression of transforming growth factor-α and epidermal growth factor receptor proteins in the human pancreas and digestive tract

This study was designed to localize transforming growth factor alpha (TGF-) and epidermal growth factor receptor (EGFR) expression in the developing human gastrointestinal tract and pancreas. Immunohistochemical techniques using specific antibodies against human TGF- and EGFR were performed on digestive tissues of fetuses from 9 to 10 to 24 weeks of gestation, children and adults. In fetuses, TGF- and EGFR proteins were expressed in all epithelial tissues studied with a good correlation and from an age as early as 9 to 10 weeks of gestation, except for TGF- in the esophagus. The strongest TGF- immunostaining was noted in the stomach and the proximal colon. Unexpectedly, immunoreactive gut endocrine cells were observed with the two antibodies used. Relatively numerous in fetuses, they decreased in number with age and were rare in adults particularly along the colon. Enteroglucagon-secreting cells were shown to express TGF- while some gastrin, somatostatin and pancreatic glucagon cells were immunostained with EGFR antibodies. The presence of TGF- and of its recetor in digestive tract epithelium and pancreatic tissues early in fetal life suggests a functional role for TGF- during the developmental process of the digestive system. We demonstrate that TGF- is also produced by endocrine cells and might have an additional mode of action other than paracrine, at least during fetal life.     

Immunohistochemical localization of transforming growth factor-α and epidermal growth factor-receptor in the mesonephros and metanephros of the chicken

Summary Transforming growth factor-alpha (TGF-) is a polypeptide related to epidermal growth factor (EGF). Both bind to EGF-receptor (EGF-R) to carry out their function in a variety of tissues and cell lines. Several studies have shown their presence in mammalian kidney, however, nothing has to date been stated concerning their existence in avian kidney. Expression of TGF- and EGF-R is reported here for the first time during the development of the chicken kidney. Using immunohistochemical techniques, we identified a TGF- (but not EGF) in mesonephric distal tubule cells from day 8 to day 20 of embryonic development and in metanephric distal tubule cells from day 14 of embryonic development to the adult. The histochemical characteristics of these cells and their histological localization suggest that they may be the principal cells of the distal tubules. Similarly, EGF-R was found in mesonephric proximal tubule cells from day 7 to day 18 of embryonic development and in metanephric proximal tubule cells from day 13 of embryonic development up to adult stages. The coexistence of both TGF- and EGF-R from the onset of development of mesonephros and metanephros supports their possible role in mechanisms of proliferation and differentiation of the cells of these organs.     

Effects of transforming growth factor β, tumor necrosis factor α and interferon γ on pancreatic islet β-cell responsiveness to transforming growth factor α

The insulin-producing pancreatic islet -cell, characterized by low proliferative potential, is normally not responsive to the polypeptide epidermal growth factor (EGF) or its homolog transforming growth factor (TGF-). Since EGF receptors in other tissues can be up-regulated by other growth factors and by cytokines, we have in this paper investigated whether such a -cell responsiveness to TGF-, or EGF, can be conferred by co-culture with interferon (IFN-), tumor necrosis factor (TNF-) or transforming growth factor (TGF-) in various combinations. To this end, fetal rat pancreatic islets enriched in -cells were isolated and cultured for 3 days with or without 200 pM or 20 nM TGF-. It was found that neither of these TGF- concentrations affected -cell mitogenesis, insulin content or insulin secretion. However, IFN- (1000 U/ml) evoked a modest stimulation of -cell replication, while suppressing insulin secretion and leaving the islet insulin content unaltered. TNF- (1000 U/ml), on the other hand, affected none of these parameters either alone or in any combination with TGF- or IFN-. However, when TNF- or IFN-, either alone or in combination, were combined with the cytokine interleukin-1, this resulted in islet disintegration, whereas the latter cytokine alone did not exert any gross necrotic changes evident by light microscopy. TGF- (500 pM) stimulated insulin secretion but did not influence islet insulin content or -cell mitogenesis either alone or in combination with TGF- (200 pM or 20 nM). In no instance could any mitogenic or secretory response to low or high concentrations of TGF- be conferred by IFN-, TNF- or TGF- whether used alone or in combinations. Hence, responsiveness to TGF- or EGF in the -cell obviously cannot be achieved by any of these peptides.Abbreviations EGF epidermal growth factor - IFN- interferon - TGF- transforming growth factor - TGF- transforming growth factor - TNF- tumor necrosis factor      

Immunolocalization of interleukin-1α in rat mandibular molars and its enhancement after in vivo injection of epidermal growth factor

Immunolocalization of interleukin-1 in the first mandibular molars of rats from day 0–12 postnatally showed that the protein was localized in the epithelial stellate reticulum adjacent to the dental follicle. Staining of the stellate reticulum was most prominent in the early days postnatally and was absent by postnatal day 11. Injection of epidermal growth factor into rats at day 0 greatly increased the intensity of the staining for interleukin-1 in the stellate reticulum. Epidermal growth factor (EGF) enhanced the gene expression of interleukin-1 in stellate reticulum cells in vitro, and this study suggests there is enhanced translation of interleukin-1 messenger RNA in the stellate reticulum following EGF injection. In turn, the interleukin-1 may exert its effect on the dental follicle cells adjacent to the stellate reticulum because EGF also enhanced expression of the interleukin-1 receptor type I messenger RNA in cultured dental follicle cells as well as enhancing its expression in vivo. In view of the fact that injection of EGF will stimulate precocious eruption of teeth, its stimulus of interleukin-1 synthesis in the stellate reticulum may be the mechanism by which EGF initiates a cascade of molecular events to signal the onset of tooth eruption.     

Characterization of residual hexosaminidase activity in Sandhoff's disease using man-Chinese hamster cell hybrids

Summary To obtain information about the nature of the residual hexosaminidase activity in Sandhoff's disease, hybrid cell lines between fibroblasts from a patients with Sandhoff's disease and Chinese hamster cells were isolated.In these hybrid cell lines, a heteropolymeric isoenzyme was detected that is composed of human - and Chinese hamster hexosaminidase subunits. Due to the electrophoretic and immunological behavior of the heteropolymeric molecules in interspecies hybrids with normal fibroblasts and with cells from a patient with Sandhoff's disease, we conclude that Sandhoff cells contain an -subunit of hexosaminidase with normal characteristics.     

p53 expression and TGF-α-induced replication of hepatocytes isolated from rats exposed to the carcinogen diethylnitrosamine

Previous reports indicate that the expression of transforming growth factor (TGF-) is increased in enzyme-altered foci (EAF) arising in livers of rats treated with a carcinogen. Here we have investigated the effects of TGF- on EAF cells in vitro. Hepatocytes were isolated from rats that had received repeated treatment with diethylnitrosamine (DEN) and whose livers contained glutathione S-transferase P (GST-P)-positive EAF. Primary cultures of GST-P-positive and GST-P-negative hepatocytes were exposed to TGF-. TGF- (20–40 ng/ml) increased DNA replication in the GST-P-negative, but not in the GST-P-positive cells. Furthermore, it was shown that this effect on GST-P-negative cells could be blocked by p53 antisense oligonucleotides. We conclude that EAF hepatocytes do not respond to TGF- in vitro. This lack of response may reflect the attenuated expression of p53 in these cells. These data corroborate previous findings that, in response to DNA damage, many EAF hepatocytes do not accumulate p53.     

Identification of human milk α-lactalbumin as a cell growth inhibitor

Summary A growth inhibitory protein, mammary inhibitory activity (MIA), was purified to apparent homogeneity from human milk. At concentrations of 5 to 10 ng/ml, the factor inhibited the growth of mammary epithelial cells by 30–80% and also inhibited the growth of normal rat kidney cells. Whereas the cell division of normal human mammary epithelium in primary culture was inhibited by MIA, cell division by fibroblasts from the same tissues was unresponsive. Inhibition was dose and time dependent and readily reversed when MIA was removed. MIA also inhibited growth in culture for three cell lines. The growth inhibitory protein migrated as a 14 kDa protein under reducing conditions on polyacrylamide gels in the presence of sodium dodecyl sulfate. The apparent isoelectric point was pI 5.0. The amino acid composition of MIA resembled that of -lactalbumin, and sequence analysis of the N-terminal region comprising residues 1–24 and an isolated peptide were identical with the N-terminal and residues 66–81 of human -lactalbumin. In addition, MIA was active in the lactose synthase system. The results strongly suggest that MIA and -lactalbumin are identical proteins. Consistent with these results, -lactalbumin preparations from several mammalian species, including human, goat, cow and camel, were all found to be growth inhibitory for cultured mammary epithelial cells. The inhibitory activity associated with human -lactalbumin was destroyed by digestion with pepsin or chymotrypsin, by carboxymethylation of cysteine, or by cleavage of methionine 90 following cyanogen bromide treatment. The results raise the possibility that during lactation -lactalbumin, a product of mammary cell differentiation, could be a physiologically relevant feed-back inhibitor of mammary cell growth and perhaps of other cell types as well.Abbreviations MIA mammary inhibitory activity - MDGI mammary derived growth inhibitor - -LA alpha lactalbumin - H--LA human -lactalbumin - NRK normal rat kidney - IMEM improved minimal essential medium - DMEM Dulbecco's modified Eagles medium - FCS fetal calf serum - EGF epidermal growth factor - TGF transforming growth factor - CNBr cyanogen bromide - SDS sodium dodecyl sulfate - kDa kilodaltons - ND-PAGE non-denaturing polyacrylamide gel electrophoresis - TCA trichloroacetic acid     

Immunolocalization of transforming growth factor alpha in normal human tissues

Transforming growth factor alpha (TGF-) is a polypeptide with well-characterized growth promoting properties. The effects are exerted through the epidermal growth factor receptor (EGF receptor), which is present on many different kinds of cells. The growth factor was initially shown to induce anchorage-independent growth of normal cells and was, therefore, considered as an oncogenic growth factor. Later, its immunohistochemical presence in normal human cells as well as its biological effects in normal human tissues have been demonstrated. The aim of the present investigation was to elucidate the distribution of the growth factor in a broad spectrum of normal human tissues. Indirect immunoenzymatic staining methods were used. The polypeptide was detected with a polyclonal as well as a monoclonal antibody. The polyclonal and monoclonal antibodies demonstrated almost identical immunoreactivity. TGF- was found to be widely distributed in cells of normal human tissues derived from all three germ layers, most often in differentiated cells. In epithelial cells, three different kinds of staining patterns were observed, either diffuse cytoplasmic, cytoplasmic in the basal parts of the cells, or distinctly localized adjacent to the nucleus, usually on the luminal aspect, corresponding to the localization of the Golgi complex. The latter staining pattern was seen predominantly in secretory epithelial cells. The present study thus confirms previous studies and elaborates new localizations of TGF- in normal human tissues by investigating a broad spectrum of tissues in detail.     

Utilization and stability of vitamins in serum-containing and serum-free media in CHO cell culture

The concentrations of four vitamins, ascorbic acid, nicotinamide, choline and thiamine were evaluated in the culture supernatant of Chinese hamster ovary (CHO) cells. The media used were -modified Eagle's minimum essential medium (MEM-) supplemented with 10% fetal calf serum, and a 1:1 mixture of Ham's F12 and Dulbecco's modified Eagle's medium (DME/F12), containing neither serum nor protein. The reference experiment without cells revealed instability of ascorbic acid and thiamine. Moreover, a significant amount of each vitamin decreased in the culture supernatant. The possibility of growth limitation by vitamin depletion is strongly suggested.     

Characterization of α-galactosidase isoenzymes in normal and fabry human-Chinese hamster somatic cell hybrids

Summary The -galactosidases in normal man-Chinese hamster somatic cell hybrids were investigated with antibodies specific for human -galactosidase A and antibodies specific for Chinese hamster -galactosidase. It was found that an isoenzyme in hybrid cells, which has an electrophoretic mobility between that of human -galactosidase A and Chinese hamster -galactosidase, contains immunologic determinants of both human and Chinese hamster origin, suggesting that it is a heteropolymeric molecule. Moreover, the locus for human -galactosidase, which was found to be X-linked, is the locus coding for -galactosidase A. Hybrids isolated after fusion of Chinese hamster cells with cells of a patient with Fabry's disease did not express human -galactosidase A or the heteropolymeric molecule even in the presence of the active human X chromosome, indicating that the deficiency of -galactosidase A in Fabry's disease is probably due to a mutation in a structural gene resulting in the inability to form immunologically detectable and functionally active molecules of -galactosidase A.     

Amplification and expression of the TGF-α, EGF receptor and c-myc genes in four human oesophageal squamous cell carcinoma lines

We have previously shown that four human oesophageal squamous cell carcinoma (SCC) cell lines secrete significant quantities of transforming growth factor alpha (TGF-)in vitro. Three of these lines are known to produce supernumary low-affinity epidermal growth factor receptors (EGF-Rs). Using an¹²⁵I-EGF compeitive binding assay and Scatchard analysis, we show that the fourth also overproduces low-affinity receptors. According to slot blot DNA analyses, the secretion of high levels of TGF- is not associated with amplification of the TGF- gene, and hyperproduction of the EGF-R is correlated with receptor gene amplification. Western blot analyses show that the c-Myc protein is overexpressed in two of the cell lines; and Southern and Northern blot analyses indicate that this overexpression occurs independently of c-myc gene amplification. Our results are consistent with an autocrine role for TGF- and EGF-R in oesophageal carcinogenesis and support the possibility that c-myc overexpression may be required for thein vivo tumourigenicity of cells that produce high levels of TGF- and the EGF-R.     

Regeneration of Lycium barbarum L. plants from leaf tissue,callus culture and callus protoplasts

The possibility of plant regeneration from leaf tissue, callus and callus protoplasts of Lycium barbarum L. has been studied. Leaf segments were cultured on B5 medium (Gamborg et al. 1968) containing 1.5 mg/1 6-benzylaminopurine and 0.5 mg/1 -naphthaleneacetic acid. Regeneration of shoots was initiated after 30 days of cultivation. Callus was obtained from leaf and internode tissues on MS medium (Murashige and Skoog 1962) containing 0.4 mg/1 of 2,4dichlorophenoxyacetic acid. Subsequently, callus was successfully subcultured on the same medium with 1 mg/l of 2,4-dichlorophenoxyacetic acid and 0.2 mg/l -naphthaleneacetic acid. Organogenesis in callus culture was obtained in the course of 40 days after transferring to TM-4 (Shahin 1984). Protoplasts were isolated from callus tissue grown in vitro using an enzymatic method. Cell colonies, minicallus formation and organogenesis were obtained. Shoots were rooted on Murashige and Skoog medium containing 0..1 mg/l -naphthaleneacetic acid. Regenerated plants were transferred to soil and were grown to maturity. Regenerated plants carried normal morphological traits.Abbreviations BA 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - Zea zeatin - GA₃ gibberellic acid - MS Murashige and Skoog medium - B5 Gamborg medium     

Expression of receptor protein tyrosine phosphatase agr; mRNA in human prostate cancer cell lines

Receptor protein tyrosine phosphatase (RPTP) is transmembrane protein phosphatases, and has been proposed to be involved in the differentiation of the neuronal system. In the present study, we demonstrated the expression of RPTP mRNA in several normal human tissues. We further investigated the regulation of expression of RPTP mRNA in epithelial cells utilizing three commercially available human prostate cancer cell lines LNCaP, PC-3 and DU145. This is because these cells exhibit different levels of differentiation, defined by the expression of a tissue-specific differentiation antigen, prostatic acid phosphatase (PAcP), and their androgen sensitivity. LNCaP cells express PAcP and are androgen-sensitive cells, while PC-3 and DU145 cells do not express PAcP and are androgen-insensitive cells. Northern blot analyses revealed that, in LNCaP cells, fetal bovine serum (FBS) and 5-dihydrotestosterone (DHT) down-regulates RPTP mRNA expression, similar to the effect on PAcP. Contrarily, FBS up-regulated the RPTP mRNA level in PC-3 and DU145 cells. In LNCaP cells, sodium butyrate inhibited cell growth and up-regulated RPTP as well as PAcP mRNA expression. Although, sodium butyrate also inhibited the growth of PC-3 and DU145 cells, the level of RPTP mRNA was decreased in PC-3, while increased in DU145 cells. Thus, data taken together indicate that the expression of RPTP is apparently regulated by a similar mechanism to that of PAcP in LNCaP cells.     

A new three-dimensional culture of human keratinocytes: optimization of differentiation

Many attempts have been made to obtain reconstructed human epidermis comprised of keratinocytes and extracellular-matrix constituents (essentially collagen) in the presence or absence of fibroblasts. A simple model of cultured human keratinocytes grown at the air-liquid interface of a noncoated artificial membrane, has been developed. This culture system offers many advantages: easy control of environmental factors and routine examination using optical or electronic microscopy, immunohistochemistry and indirect immunofluorescence techniques. This model enables the analysis of well-known differentiation markers and also intregrins, a family of cell-surface molecules involved in cell-cell and cell-extracellular matrix interactions, whose receptors are expressed on all basal keratinocytes.In our culture system, the expression of the different integrin subunits (2, 3, 5, 6, 1) was studied as a function of the differentiation state in two different media (K-SFM or DMEM/Ham's F12) supplemented with 5% fetal calf serum and adjusted to 1.5 mmol/L calcium. The most significant data are the preponderant expression of the 2 and 3 subunits in the basal and suprabasal layers, with membrane expression differing according to the culture medium; terminal differentiation was obtained in DMEM/Ham's F12. The use of membrane inserts represents a significant technological advance in culturing keratinocytes and is an easy-to-handle and valid model for determining the influence of physiological or pharmacological factors on cell proliferation or differentiation.Abbreviations DMEM Dulbecco's Modified Eagle's Medium - EGF epidermal growth factor - FCS fetal calf serum - K-SFM keratinocyte serum free medium - MAb monoclonal antibody - NHEK normal human epidermal keratinocytes - PBS Dulbecco's phosphate-buffered saline     

Stable expression in Chinese hamster ovary cells of a homogeneous recombinant active fragment of human platelet glycoprotein Ibα

A fragment (residues His1-Val289) of the chain of human platelet glycoprotein Ib containing the von Willebrand factor and thrombin binding sites has been expressed in Chinese hamster ovary cells. The secreted soluble recombinant protein had an apparent molecular mass of 42 kD and reacted with a conformation-dependent monoclonal antibody that only binds to native GP Ib, thus demonstrating its proper folding. The rather broad band obtained after immobilization of the recombinant fragment on nitrocellulose could be resolved into a very sharp band of molecular weight of about 35 kD by growing the cells in the presence of tunicamycin, and inhibitor of N-linked glycosylation. The recombinant GP Ib fragments (with or without glycosylation) were purified by immunoaffinity chromatography. Both truncated forms bound vWF in the presence of botrocetin with comparable affinity as a proteolytic 42 kD fragment of purified human platelet GP Ib-IX. They were also retained on thrombin-Sepharose. We then selected a cell clone (B1) that produced over at least three months about 1.5 g of recombinant protein per million cells per day. Using this clone a large-scale production finally yielded milligram amounts of the functionally active recombinant human GP Ib fragment.Abbreviations ABTS 2.2-azino-di-(3-ethylbenzthiazoline sulphonate) - CHO Chinese hamster ovary - dhfr dihydrofolate reductase - GP Ib-IX glycoprotein Ib-IX complex - HEPES 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid - IEF isoelectric focusing - Ig immunoglobulin - mAb monoclonal antibody - MEM minimum essential medium - PMSF phenyl-methylsulfonyl fluoride - SDS sodium dodecyl sulfate - vWF von Willebrand factor