bluuebunny/crossref_metadata_2025 huggingface dataset:
Expression of estrogen receptors in estrogen receptor–negative human breast carcinoma cells: Modulation of epidermal growth factor‐receptor (EGF‐R) and transforming growth factor α (TGFα) gene expression
Authors: Sheikh M. Saeed , Shao Zhi‐Ming , Chen Jian‐Chyi , Li Xiao‐Su , Hussain Arif , Fontana Joseph A.
Published: May 2005

A number of studies suggest that an inverse correlation exists between the epidermal growth factor–receptor and the estrogen receptor expression in primary human breast carcinoma as well as in established human breast carcinoma cell lines. Recent studies suggest that the epidermal growth factor–receptor does not regulate the estrogen receptor gene expression. Whether the estrogen receptor regulates the epidermal growth factor–receptor gene expression is not known. We addressed this question by stably transfecting the estrogen receptor cDNA into the estrogen receptor–negative human breast carcinoma cell line MDA‐MB‐231. Constitutive expression of functional estrogen receptors in the transfactants resulted in increased mRNA levels of both epidermal growth factor–receptor and transforming growth factor α. Estradiol treatment of transfected cells, although enhancing transforming growth factor α mRNA levels, did not modulate epidermal growth factor–receptor mRNA levels. The estrogen receptor–transfected cells grown in estrogenic regular medium, however, exhibited lower constitutive levels of epidermal growth factor–receptor mRNA than in steroid‐stripped medium, suggesting that estrogens coupled with some factors normally present in the regular medium may indeed downmodulate epidermal growth factor–receptor mRNA. Sodium butyrate treatment enhanced epidermal growth factor–receptor mRNA levels in nontransfected cells grown in regular estrogenic as well as in steroid stripped medium. Sodium butyrate enhancement of epidermal growth factor–receptor mRNA levels was completely abolished in estrogen receptor–transfected cells grown in regular estrogenic medium and blunted in steroid stripped medium. Using various epidermal growth factor–receptor gene promoter‐CAT constructs in transient transfection assays, we further demonstrate that sodium butyrate enhanced transcription of the epidermal growth factor–receptor gene. The putative sodium butyrate responsive element(s) appears to localize within the proximal 384 bp of the epidermal growth factor–receptor gene promoter region. Although the interactions between estrogen receptor and epidermal growth factor–receptor are rather complex, taken together, our data suggest that estrogen receptor can indeed modulate the epidermal growth factor–receptor mRNA expression.
Apolipoprotein E: A potent inhibitor of endothelial and tumor cell proliferation
Authors: Vogel Tikva , Guo Neng‐Hua , Guy Rachel , Drezlich Nina , Krutzsch Henry C. , Blake Diane A. , Panet Amos , Roberts David D.
Published: May 2005

Recombinant human apolipoprotein E3 (apoE), purified from E. coli, inhibited the proliferation of several cell types, including endothelial cells and tumor cells in a dose‐ and time‐dependent manner. ApoE inhibited both de novo DNA synthesis and proliferation as assessed by an increase in cell number. Maximal inhibition of cell growth by apoE was achieved under conditions where proliferation was dependent on heparin‐binding growth factors. Thus, at low serum concentrations (0–2.5%) basic fibroblast growth factor (bFGF) stimulated the proliferation of bovine aortic endothelial (BAE) cells severalfold. The bFGF‐dependent proliferation was dramatically inhibited by apoE with an IC50 ≈ 50 nM. Under conditions where cell proliferation was mainly serum‐dependent, apoE also suppressed growth but required higher concentrations to be effective (IC50 ≈ 500 nM). ApoE also inhibited growth of bovine corneal endothelial cells, human melanoma cells, and human breast carcinoma cells. The IC50 values obtained with these cells were generally 3–5 times higher than with BAE cells. Inhibition of cell proliferation by apoE was reversible and dependent on the time of apoE addition to the culture. In addition, apoE inhibited the chemotactic response of endothelial cells that were induced to migrate by a gradient of soluble bFGF. Inhibition of cell proliferation by apoE may be mediated both by competition for growth factor binding to proteoglycans and by an antiadhesive activity of apoE. The present results demonstrate that apoE is a potent inhibitor of proliferation of several cell types and suggest that apoE may be effective in modulating angiogenesis, tumor cell growth, and metastasis.
Induction of cell surface peptidase activity: A global response to cell stress correlated with apoptosis
Authors: Brown S. B. , Kluck R. M. , Ellem K. A. O.
Published: May 2005

We have previously characterized the stimulation of HeLa cell surface peptidase activity directed toward a nonapeptide substrate in response to low fluences of ultraviolet irradiation [Brown et al. (1993): J Cell Biochem 51:102–115]. To explore the hypothesis that this comprised a global response to cell stress featuring the interruption of DNA synthesis, a variety of agents affecting macromolecular synthesis were applied to HeLa cell cultures. Actinomycin D, 5,6‐dichloro‐1 β‐ribofuranosyl benzimadazole, mitomycin C, ultraviolet light, and cycloheximide at doses which inhibited cell growth, but fell short of increasing the proportion of cells which had lost cell membrane impermeability to trypan blue, resulted in the concentration dependent increase in both amino‐ and endo‐peptidase activities of intact HeLa cell cultures. γ‐Irradiation, despite inhibiting an increase in cell number over a 20‐h observation period, had no effect on the expressed level of cell surface peptidase activity nor did the accumulation of cells in S or G2 phase by thymidine parasynchronization. Some of these agents were found to increase the proportion of cells in the culture undergoing apoptosis (programmed cell death), and a strong correlation was found between the extent of apoptosis and the degree of elevation in cell surface peptidase activity. Higher concentrations of perturbants in some instances increased the percentage of cells that were nonviable and an associated release of intracellular proteases overwhelmed the linear correlation with apoptotic cells. The present data do not distinguish between a homogeneous elevation of surface peptidase activity in all cells of treated cultures or the heterogeneous increase in only preapoptotic or apoptotic cells. Since sunburn of the skin increases both the occurrence of apoptotic keratinocytes (sunburn cells) in the affected epidermis and the release of membrane bound cell activators such as transforming growth factor α, it is suggested by way of extrapolation of these in vitro results, that the increase in cell surface proteolytic activity plays an integral part in the reparative responses of the epidermal cells in vivo.
Interleukin 4 increases type 5 acid phosphatase mrna expression in murine bone marrow macrophages
Authors: Lacey D. L. , Erdmann J. M. , Tan H.‐L.
Published: May 2005

Type 5 acid phosphatase is a lysosomal enzyme expressed in cells of monocyte/macrophage lineage frequently used as a marker of osteoclastic differentiation. Oligonucleotide primers for DNA amplification were designed following sequence alignment of rat bone and human macrophage type 5 acid phosphatases. DNA (330 bp in length) obtained using these primers and reverse transcribed total cell RNA from in vitro generated murine osteoclastic cells was cloned and sequenced. DNA sequence analysis of two clones demonstrates that the amplified material was 91% and 96% identical to rat bone type 5 acid phosphatase at the nucleotide and amino acid level, respectively. Northern blots of murine tissue RNA show the presence of 1.5‐kb transcripts that are most highly expressed in the long bones. Total cell RNA from the osteoclastic cells contain a marked level of type 5 acid phosphatase mRNA when compared to the levels seen in the tissue samples. Additionally, osteoclastic cell RNA contains two additional transcripts of 2.5 and 5 kb. Bone marrow macrophages grown in the presence of M‐CSF express low levels of the 1.5‐kb transcript with no signal observed for either of the two larger transcripts that were seen in the osteoclastic RNA samples. Importantly, bone marrow macrophage 1.5‐kb type 5 acid phosphatase transcript levels are increased by interleukin 4 treatment in both a time and concentration‐dependent manner. These findings indicate that type 5 acid phosphatase, while a cytochemical marker for osteoclasts, can be induced in macrophages by agents that block in vitro osteoclastic differentiation. Increased type 5 acid phosphatase may play a role in interleukin 4‐stimulated monocyte activities.
Posttranscriptional regulation of thymidylate synthase gene expression
Authors: Johnson Lee F.
Published: May 2005

Thymidylate synthase (TS) is an essential enzyme that catalyzes the formation of thymidylic acid in the de novo biosynthetic pathway and is the target enzyme for a variety of chemotherapeutic agents. The TS gene is expressed at a much higher level in proliferating cells than in quiescent cells. Control is primarily exerted at the posttranscriptional level. Studies with chimeric TS minigenes have shown that regulation of TS mRNA content in growth‐stimulated mouse fibroblasts requires the presence of sequences located upstream of the essential promoter elements. In addition, an efficiently spliced intron must be present within the transcript. Neither sequence by itself is sufficient for proper regulation, suggesting that the upstream and downstream sequences may communicate to effect regulation. A possible mechanism by which the upstream sequences influence the efficiency of splicing of TS transcripts in a cell cycle specific manner is described.

Expression of the human TS gene is also controlled at the translational level. The TS enzyme is able to block the translation of its own mRNA by binding to the message in the vicinity of the AUG start codon. The translational block is relieved in the presence of substrates or inhibitors of the enzyme. The autogenous translational regulation of TS mRNA is likely to be responsible for the rapid increase in TS enzyme level that occurs when cells are exposed to certain TS inhibitors. Elucidation of the mechanism by which the translational control is exerted may lead to the design of more effective TS inhibitors.
Control of fibroblast senescence and activation of programmed cell death
Authors: Wang Eugenia , Lee Menq‐Jer , Pandey Siyaram
Published: May 2005

We have characterized a nuclear phosphoprotein of 57 kda, statin, found only in nonproliferating cells of both quiescent and senescent natures. Emerging results suggest that statin may function as a sequester to block the early G1 phase phosphorylation for the RB protein. A second protein, terminin, undergoes senescence‐specific posttranslational modification from 90 to 60 kda, and further death‐specific conversion from 60 to 30 kda. We also found that apoptotic mouse 3T3 fibroblasts express c‐fos, c‐myc, c‐jun, and cdc2, as well as the upregulation of RB phosphorylation and BrdU incorporation, just before final DNA fragmentation and death. It seems that en route to death, cells re‐enter the cell‐cycle traverse and experience early G1 and part of S Phase; however, this cycling event is an abortive one. In contrast, senescent fibroblasts are resistant to the initiation of the death program, since they are unable to enter cell cycle traverse. Long‐term serial passaging of normal human fibroblasts may be inadvertently selecting those, while termed as senescent, are also specialized survivors, and thus a good culture model to study both the control of permanent departure from cell cycle traverse and the mechanism underlying the survival or antideath cellular program.
Cell cycle control and cancer chemotherapy
Authors: Kohn Kurt W. , Jackman Joany , O'Connor Patrick M.
Published: May 2005

As detailed information accumulates about how cell cycle events are regulated, we can expect new opportunities for application to cancer therapy. The altered expression of oncogenes and tumor suppressor genes that commonly occurs in human cancers may impair the ability of the cells to respond to metabolic perturbations or stress. Impaired cell cycle regulation would make cells vulnerable to pharmacologic intervention by drug regimens tailored to the defects existing in particular tumors. Recent findings that may become applicable to therapy are reviewed, and the possible form of new therapeutic stratagems is considered.
Cell density‐dependent regulation of cell surface expression of two types of human tumor necrosis factor receptors and its effect on cellular response
Authors: Pocsik Eva , Mihalik Rudolf , Ali‐Osman Francis , Aggarwal Bharat B.
Published: May 2005

Tumor necrosis factor (TNF) is a multipotential cytokine known to regulate the growth of a wide variety of normal and tumor cells. It has been shown that the density of cells in culture can modulate the growth regulatory activities of TNF, the mechanism of which, however, is not understood. In this report, we investigated the effect of cell density on the expression of TNF receptors. The receptors were examined on epithelial cells (e.g., HeLa), which primarily express the p60 form, and on myeloid cells (e.g., HL‐60) known to express mainly the p80 form. We observed that binding of TNF to both cell lines decreased with increase in cell density. Scatchard analysis of binding on HeLa and HL‐60 cells revealed a 4‐ to 5‐fold reduction in the number of TNF receptors without any significant change in receptor affinity in both cell types at high density. The decrease in TNF receptor numbers at high cell density was also observed in several other epithelial and myeloid cell lines. The downmodulation at high cell density was unique to TNF receptors, since minimum change in other cell surface proteins was observed as revealed by fluorescent activated cell sorter analysis. Neutralization of binding with antibodies specific to each type of the receptors revealed that both the p60 and p80 forms of the TNF receptor were equally downmodulated.

A decrease in leucine incorporation into proteins was observed with increase in cell density, suggesting a reduction in protein synthesis. Since inhibition of protein synthesis by cycloheximide also leads to a decrease in TNF receptors, it is possible that the density‐dependent reduction in TNF receptor number is due to an overall decrease in protein synthesis. The density‐dependent decrease in TNF receptors was accompanied by a decrease in intracellular reduced glutathione levels. A reduction in the number of receptors on TNF sensitive tumor cells induced by cell‐density correlated with increase in resistance to the cytokine.
Nuclear architecture supports integration of physiological regulatory signals for transcription of cell growth and tissue‐specific genes during osteoblast differentiation
Authors: Stein Gary S. , van Wijnen André J. , Stein Janet L. , Lian Jane B. , Bidwell Joseph P. , Montecino Martin
Published: May 2005

During the past several years it has become increasingly evident that the three‐dimensional organization of the nucleus plays a critical role in transcriptional control. The principal theme of this prospect will be the contribution of nuclear structure to the regulation of gene expression as functionally related to development and maintenance of the osteoblast phenotype during establishment of bone tissue‐like organization. The contributions of nuclear structure as it regulates and is regulated by the progressive developmental expression of cell growth and bone cell related genes will be examined. We will consider signalling mechanisms that integrate the complex and interdependent responsiveness to physiological mediators of osteoblast proliferation and differentiation. The focus will be on the involvement of the nuclear matrix, chromatin structure, and nucleosome organization in transcriptional control of cell growth and bone cell related genes. Findings are presented which are consistent with involvement of nuclear structure in gene regulatory mechanisms which support osteoblast differentiation by addressing four principal questions: (1) Does the representation of nuclear matrix proteins reflect the developmental stage‐specific requirements for modifications in transcription during osteoblast differentiation? (2) Are developmental stage‐specific transcription factors components of nuclear matrix proteins? (3) Can the nuclear matrix facilitate interrelationships between physiological regulatory signals that control transcription and the integration of activities of multiple promoter regulatory elements? (4) Are alterations in gene expression and cell phenotypic properties in transformed osteoblasts and osteosarcoma cells reflected by modifications in nuclear matrix proteins? There is a striking representation of nuclear matrix proteins unique to cells, tissues as well as developmental stages of differentiation, and tissue organization. Together with selective association of regulatory molecules with the nuclear matrix in a growth and differentiation‐specific manner, there is a potential for application of nuclear matrix proteins in tumor diagnosis, assessment of tumor progression, and prognosis of therapies where properties of the transformed state of cells is modified. It is realistic to consider the utilization of nuclear matrix proteins for targeting regions of cell nuclei and specific genomic domains on the basis of developmental phenotypic properties or tissue pathology. © 1994 Wiley‐Liss, Inc.
Conformational information in DNA: Its role in the interaction with DNA topoisomerase I and nucleosomes
Authors: Caserta Micaela , Venditti Patrizia , Mauro Ernesto Di , Camilloni Giorgio , Venditti Sabrina
Published: May 2005

Information in DNA is not limited to sequence information. Both local and global conformational parameters are pivotal to the interaction with a number of relevant proteins. The function of the major components of the transcription machinery (RNA polymerase II, DNA topoisomerase I, nucleosomes, the TATA‐binding factor) is dependent on the topological status of the substrate DNA molecule. The topological requirements and the conformational consensus that dictate the rules for localization of nucleosomes and define the active sites for DNA topoisomerase I have been established; the reaction of DNA topoisomerase I is regulated by a topological feedback mechanism. The integrating function of the free energy of supercoiling in the transcription process and the regulatory role of DNA topoisomerase I are discussed. © 1994 Wiley‐Liss, Inc.
Role of vascular endothelial cells in bone biology
Authors: Collin‐Osdoby Patricia
Published: May 2005

Bone development and remodeling depend on complex interactions between bone‐forming osteoblasts, bone‐degrading osteoclasts, and other cells present within the bone microenvironment. Balanced control of bone formative and degradative processes is normally carefully maintained in the adult skeleton but becomes uncoupled in the course of aging or in various pathological disease states. Systemic regulators of bone metabolism and local mediators, including matrix molecules, cytokines, prostaglandins, leukotrienes, and other autocrine or paracrine factors, regulate the recruitment, differentiation, and function of cells participating in bone formation and turnover. Although some of these interactions are now understood, many yet remain to be elucidated. Recent studies have begun exploring in detail how vascular endothelial cells and their products function in bone physiology. The findings are revealing that bone vascular endothelial cells may be members of a complex communication network in bone which operates between endothelial cells, osteoblasts, osteoclasts, macrophages, stromal cells, and perhaps other cell types found in bone as well. Therefore, multiple systemic and locally produced signals may be received, transduced, and integrated by individual cells and then propagated by the release from these cells of further signals targeted to other members of the bone cell network. In this manner, bone cell activities may be continuously coordinated to afford concerted actions and rapid responses to physiological changes. The bone microvasculature may play a pivotal role in these processes, both in linking circulatory and local signals with cells of the bone microenvironment and in actively contributing itself to the regulation of bone cell physiology. Thus, skeletal homeostasis and the coupling observed between bone resorption and bone formation during normal bone remodeling may be manifestations of this dynamic interactive communication network, operating via diverse signals not only between osteoblasts and osteoclasts but between many cell types residing within bone. © 1994 Wiley‐Liss, Inc.
Signal transduction pathways mediating parathyroid hormone regulation of osteoblastic gene expression
Authors: Partridge Nicola C. , Bloch Sharon R. , Pearman A. Terrece
Published: May 2005

Parathyroid hormone (PTH) plays a central role in regulation of calcium metabolism. For example, excessive or inappropriate production of PTH or the related hormone, parathyroid hormone related protein (PTHrP), accounts for the majority of the causes of hypercalcemia. Both hormones act through the same receptor on the osteoblast to elicit enhanced bone resorption by the osteoclast. Thus, the osteoblast mediates the effect of PTH in the resorption process. In this process, PTH causes a change in the function and phenotype of the osteoblast from a cell involved in bone formation to one directing the process of bone resorption. In response to PTH, the osteoblast decreases collagen, alkaline phosphatase, and osteopntin expression and increases production of osteocalcin, cytokines, and neutral proteases. Many of these changes have been shown to be due to effects on mRNA abundance through either transcriptional or post‐transcriptional mechanisms. However, the signal transduction pathway for the hormone to cause these changes is not completely elucidated in any case. Binding of PTH and PTHrP to their common receptor has been shown to result in activation of protein kinases A and C and increases in intracellular calcium. The latter has not been implicated in any changes in mRNA of osteoblastic genes. On the other hand activation of PKA can mimic all the effects of PTH; protein kinase C may be involved in some responses. We will discuss possible mechanisms linking PKA and PKC activation to changes in gene expression, particularly at the nuclear level. © 1994 Wiley‐Liss, Inc.
Dual role of osteoblastic proenkephalin derived peptides in skeletal tissues
Authors: Rosen Haim , Bar‐Shavit Zvi
Published: May 2005

Proenkephalin encodes a group of small peptides with opiate‐like activity, the endogenous opioids, known to function as neurohormones, neuromodulators, and neurotransmitters. Recently, we have demonstrated that in addition to its abundance in fetal brain tissue, proenkephalin is highly expressed in nondifferentiated mesodermal cells of developing fetuses. We identified the skeletal tissues, bone, and cartilage as major sites of proenkephalin expression. To examine the possibility that proenkephalin is involved in bone development we have studied the expression of this gene in bone‐derived cells, its modulation by bone active hormones, and the effects of enkephalin‐derived peptides on osteoblastic phenotype. Our studies revealed that osteoblastic cells synthesize high levels of proenkephalin mRNA which are translated, and the derived peptides are secreted. Reciprocal interrelationships between osteoblast maturation and proenkephalin expression were established. These results together with our observations demonstrating inhibitory effects of proenkephalin‐derived peptides on osteoblastic alkaline phosphatase activity, strongly support the notion that proenkephalin is involved in bone development. A different direction of research by other investigators has established the capability of the opioid system in the periphery to participate in the control of pain. On the basis of these two lines of observation, we would like to present the following hypothesis: The potential of embryonic skeletal tissue to synthesize proenkephalin‐derived peptides is retained in the adult in small defined undifferentiated cell populations. This potential is realized in certain situations requiring rapid growth, such as remodeling or fracture repair. We suggest that in these processes, similarly to the situation in the embryo, the undifferentiated dividing cells produce the endogenous opioids. In the adult these peptides may have a dual function, namely participating in the control of tissue regeneration and in the control of pain. © 1994 Wiley‐Liss, Inc.
Role of colony‐stimulating factor‐1 in bone metabolism
Authors: Felix Rolf , Hofstetter Willy , Wetterwald Antoinette , Cecchini Marco G. , Fleisch Herbert
Published: May 2005

Colony‐stimulating factor‐1 (CSF‐1) is a cytokine required for proliferation, differentiation, activity, and survival of cells of the mononuclear phagocytic system. The growth factor is synthesized as a soluble, matrix, or membrane associated molecule. The specific functions of these forms are not clear. However, some data suggest a dependence of the development of various populations of tissue macrophages on the locally expressed and presented cytokine. Deficiency in CSF‐1, as is the case in the murine mutant strain op/op, results in low numbers of macrophages and monocytes and, most striking, leads to osteopetrosis due to a virtual absence of osteoclasts. Using the op/op mutation as a model, CSF‐1 was established as one of the growth factors for osteoclasts. The expression of CSF‐1 receptors, encoded by the proto‐oncogene c‐fms, by osteoclast precursors and osteoclasts, suggested an effect of this cytokine not only during osteoclast formation but also on the mature cells. In fact, CSF‐1 was shown to inhibit the resorbing activity, to stimulate migration, and to support survival of isolated osteoclasts in vitro. By these actions on cells of the osteoclast lineage, CSF‐1 induces recruitment of new osteoclasts, leading to a net increase of bone resorption, and might govern the spatial distribution of resorption sites within the bone. During these processes, locally expressed and presented forms of the growth factor may play a crucial role, as will be discussed in this article. © 1994 Wiley‐Liss, Inc.
Prostaglandin F2α activates phospholipase D independently from activation of protein kinase C in osteoblast‐like cells
Authors: Kozawa Osamu , Suzuki Atsushi , Kotoyori Jun , Tokuda Haruhiko , Watanabe Yasuko , Ito Yoshiaki , Oiso Yutaka
Published: May 2005

We previously reported that prostaglandin F2α (PGF2α) receptor is coupled to pertussis toxin (PTX)–sensitive GTP‐binding protein (G protein) in osteoblast‐like MC3T3‐E1 cells [Miwa et al. (1990): Biochem Biophys Res Commun 171:1229–1235]. In the present study, we examined the effect of PGF2α on the activation of phosphatidylcholine‐hydrolyzing phospholipase D in MC3T3‐E1 cells. PGF2α stimulated the formation of choline in a dose‐dependent manner in the range between 10 nM and 10 μM. The formation of choline was stimulated by 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), a protein kinase C (PKC)–activating phorbol ester. 4α‐Phorbol 12,13‐didecanoate, a PKC‐nonactivating phorbol ester, had little effect on choline formation. The formation of choline stimulated by a combination of PGF2α and TPA was additive. Staurosporine, an inhibitor for protein kinases, which inhibited the effect of TPA on choline formation, dose‐dependently enhanced the formation of choline induced by PGF2α. NaF, an activator of G protein, stimulated the formation of choline. The formation of choline stimulated by a combination of PGF2α and NaF was not additive. NaF‐induced formation of choline was dose‐dependently enhanced by staurosporine. PTX dose‐dependently inhibited the PGF2α‐induced formation of choline. These results strongly suggest that PGF2α activates phospholipase D independently from the activation of PKC in osteoblast‐like cells and PTX‐sensitive G protein is involved in the PGF2α‐induced phospholipase D activation. © 1994 Wiley‐Liss, Inc.
Colocalization of the p185HER2 oncoprotein and integrin α6β4 in Calu‐3 lung carcinoma cells
Authors: Campiglio Manuela , Tagliabue Elda , Srinivas Uppugunduri , Pellegrini Rita , Martignone Stefania , Ménard Sylvie , Colnaghi Maria Ines , Lombardi Luciano , Marchisio Pier Carlo
Published: May 2005

Anti‐p185HER2 monoclonal antibodies often show intense reactivity with the basement membrane of tumor cells that overexpress the HER2/neu gene product (p185HER2). To evaluate a possible interaction between p185HER2 and adhesion molecules or their receptors, the polarity of p185HER2 was tested in lung carcinoma cell line Calu‐3, which overexpresses this protein, in cultures grown as confluent monolayers or as aggregates. MAb immunostaining patterns indicated that p185HER2 is concentrated on the baso‐lateral membrane of cells and that it colocalizes with the integrin α6β4 at the cell‐cell junctions where laminin is also found. The same membrane region showed intense reactivity with antiphosphotyrosine antibodies. Furthermore, integrin clustering induced by the specific antibody was accompanied by the clustering of p185HER2, as indicated by immunoelectron microscopy, and by a subsequent increase in p185HER2 tyrosine phosphorylation. Treatment with exogenous laminin also resulted in increased basal levels of p185HER2 phosphorylation. These data suggest a physical interaction between the integrin and the oncoprotein that might be functionally relevant in directly controlling the tyrosine phosphorylation of the catalytic domain of p185HER2. © 1994 Wiley‐Liss, Inc.
Progesterone but not ras requires MPF for in vivo activation of MAPK and S6 KII: MAPK is an essential conexion point of both signaling pathways
Authors: Carnero Amancio , Jiménez Benilde , Lacal Juan Carlos
Published: May 2005

Induction of mitosis in Xenopus laevis oocytes by hormones and the oncogenic ras‐p21 protein has been shown to correlate with a cascade of phosphorylations of the Ser/Thr family of kinases. However, the exact hierarchy of enzymes and their mutual interdependency has not been fully elucidated yet. We have used the Xenopus laevis system to investigate the mechanism of activation of the Ser/Thr kinases cascade and their relationship. Comparison between progesterone‐induced germinal vesicle breakdown (GVBD), a hallmark of mitosis in oocytes, to that triggered by ras‐p21, revealed the existence of at least two independent mechanisms to activate the MAP kinase enzyme in vivo. While progesterone function is dependent of cdc2 protein kinase activity, ras‐p21 is independent of this enzyme. However, both progesterone and ras‐p21 converge at the MAP kinase level, and depletion of MAP kinase activity inhibits the GVBD and S6 kinase II activation induced by both progesterone and ras‐p21. These results provides further evidence that MAP kinase is a critical step for regulation of the cell cycle in oocytes and a critical point where ras and progesterone signaling converge. © 1994 Wiley‐Liss, Inc.
Human breast and colon cancers exhibit alterations of DNA methylation patterns at several DNA segments on chromosomes 11p and 17p
Authors: Ribieras S. , Song‐Wang X.‐G. , Martin V. , Lointier P. , Frappart L. , Dante R.
Published: May 2005

In breast and colon adenocarcinomas methylation patterns at CCGG sites of several loci located on the short arm of chromosome 11 were determined by Southern blot analysis. Results obtained indicate that all tumor samples (20/20) exihibit DNA methylation changes when compared to their normal counterparts. In colon tumors,i‐globin gene is usually hypomethylated (9/10), whereas Ha‐ras gene, which is located in the same region, retains an unmodified DNA methylation pattern. Hypomethylation of parathyroid hormone (5/10) and catalase genes (4/10) are also frequently detected in colon tumor specimens. For the catalase gene the region around exon is the only one which is affected by these changes. In breast adenocacinoma, modifications of the methylation patterns are less frequently observed. However, hypomethylation of the i‐globin gene is a very common event in these tumors (8/10), and it is also detected (2/2) in lobular carcinoma in situ which is an early step in breast tumorgenesis. In addition, hypermethylation of a CpG island is also observed at the locus 17p13.3 in both colon (5/5) and breast (4/9) adenocarcinomas. In the tumoral tissues which containj a limited amount of CpG. Some of these alterations seen, therefore, to be tumor and sequence specific.
Manipulation of cellular interactions with biomaterials toward a therapeutic outcome: A perspective
Authors: Pierschbacher Michael D. , Polarek James W. , Craig William S. , Tschopp Juerg F. , Sipes Nancy J. , Harper John R.
Published: May 2005

Manipulation of the Wound healing process and the manner in which tissues interact with inertbiomaterials were both made possible with the discovery of arginine‐glucine (RGD) acid as a major cell recognition signal in the extracellular matrix. Whether promoting cell adhesion can be rationally designed to incorporate both stability and integrin specificity. Synthetic peptides containing this sequence have been linked to biodegradable biopolumers and introduced for the enhancement of dermal and corneal wound healing. By accelerating the healing reaction using RGD‐containing peptides, the quality of regenerted tissue seems to be improved, the extent of fibrosis retricted, and the risk of microbial infection may be reduced. Controlling the degree of fibrosis that often accmmpanies the healing of wounds and the reaction of tissue to foreign materials can also be achieved by natural antagonists of fibrogenic activity of TGF‐beta animal models of kidney fobrosis. There advances in the biotechnology of wound healing and tissue regeneration eventually will have an overal impact on the quality of health care.
Recent progress in immunoisolated cell therapy
Authors: Lysaght Michael J. , Frydel Beata , Gentile Frank , Emerich Dwaine , Winn Shelley
Published: May 2005

Biohybrid implalts represent a new class of medical device in which living cells, supported in a hydrogel matrix, and surrounded by semipepmiable membrane, produce and deliver therapeutic reagents tm specific sites wthin a host. First prpmsed in the mid‐1970s for tpeatment modality has progressed rapidly in the past four years and is now being investigated not just for endcrine disorders but also for alleviation of chronic pain, treatment of neurodegenerative disorders, and delivery of neurotrophic factors to sites withil the blood brain barrier, and aq a practical alternative to conventional ex vivo.
Oscillatory behavior of control‐systems of calcium homeostasis in chickens
Authors: Hurwitz S. , Miller Barbara , Norman A. W.
Published: May 2005

Computer simulation of calcium homeostasis in chicks predicted an oscillatory behavior of bone calcium flow and kidney 25‐hydroxyvitamin D3‐1 hydroxylase with a periodicity of 56 h and a 9 h phase difference between the two signals. In growing chickens subjected to a light: dark cycle of 22:2 h, and intravenously dosed with 45Ca, the temporal changes in plasma 45Ca could be described by an exponential decline with superimposed diurnal oscillations. The activity of the renal 25‐hydpoxyvitamil D3‐1‐hydroxylase in chicks subjected to a 12:12 h light: dark cycle ALSO followed diupnal oscillations, with a ladir at the beginning of the light period and a peak 12 h later. The production of 1,25‐dihydroxyvitamin D3 by primary cultures of chicken kidney cells mscillated with a periodicity of 5.6 h or shorter. It is suggested that despite the differences in phase and periodicity between the simulation predictions and actual results, the oscillations in both 1‐hydroxylase and bone calcium flow could be coupled through the hormonal systems involved in regulation of plasma calcium.
Expression of cell growth and bone phenotypic genes during the cell cycle of normal diploid osteoblasts and osteosarcoma cells
Authors: McCabe Laura R. , Last T. J. , Lynch Maureen , Lian Jane , Stein Janet , Stein Gary
Published: May 2005

Establishing reuglatory mechanisms that mediate proliferation of osteoblasts while restricting expression of genes asociated with mature bone cell phenotypic properties to post‐proliferative cells is fundamental to understanding skeletal development. To gain insight into relationships between growth control and the developmental expression of genes during osteblast differentiation, we have examined expression of three classes of genes during the cell cycle of normal diploid rat calvarial‐derived osteoblasts and rat osteosarcoma cells (ROS 17/2.8): cell cycle and growth‐related to the biosynthesis, organization, and mineralization of the bone extracellular matrix (e.g., alkaline phosphatase, collagen l, osteocalcin, and osteopontin). In normal diploid osteoblasts as well as in osteosarcoma cells we found that histone genes, required for cell progression, are selectively expressed during S phase. All other genes studied were constitutively expressed both at the transcriptional and posttranscriptional levels. Alkaline phosphatase, an integral membrane protein in both osteoblasts and osteosarcoma cells, exhibited only minimal changes in activity during the osteoblast and osteosarcoma cell cycles. Our findings clearly indicate that despite the loss of normal proliferation‐differentiation interrelationships in osteosarcoma cells, cell cycle regulatin or constitutive expression of growth and phenotypic genes is maintained.
Nongenomic actions of the steroid hormone 1α25‐dihydroxyvitamin D3
Authors: Baran Daniel T.
Published: May 2005

Recent studies indicate that the vitamin D hormone, 1α,25‐Dohydroxyvitamin D3 exerts rapid effects (seconds to minutes) in a variety of cell types. These rapid nongenomic actions in osteoblasts include effects on membrance voltage‐gated calcium chananels, phosphlipase C activity, and the sodium/dydrogen antiport. Since the rapid effects occur in osteoblasts that lack the neclear vitamin D receptor, it is postulated that the nongenomic responses to the hormone reflect interaction with a separate, membrane localized signalling system. Preliminary studies demonstrate the presence of a receptor on the membranes of osteoblasts that lack the neclear vitamin D. This membranes receptors recognizes 1 a, 25‐dihyrooxyvitamin D3 and its inaction 1β epimer, but not 25‐hydrovitamin D3. These rapid nongenomic actions generated by interaction with the membrane receptor modulate the effect of the hormone on gene transcription. Thus, the rapid nongenomic pathway may play a regulatory function in modulating the genomic pathways affected by 1 a 25‐dihydroxyvitamin D3.
Mechanisms by which cells of the osteoblast lineage control osteoclast formation and activity
Authors: Martin T. J. , Ng K. W.
Published: May 2005

The cells of bone are of two lineages, the osteoblasts arising from pluripotential mesenchymal cells and osteoclasts from hemopoietic precursors of the monocyte‐macrophage series. Resorption of bone by the multinucleate osteoclast requires the generation of new osteoclastsw and their activation. Many hormones and cytokines are able to promote bone resorption by influencing these processes, but they achieve this without acting directly on osteoclastws. Most evidence indicates that their actions are mediated by cells of the osteoblast lineage. Evidence for hormone‐and cytokine‐induced activation of osteoclasts requiring the mediation of osteoblasts comes from studies of rsorption by isolated osteoclasts. However, consistent evidence for a spiceific “activating factor” is lacking, and the argument is presented that the isolated osteoclast resorption assays have not been shown convincingly to be assays of osteoclast activation. The view is presented that osteoblast‐mediated osteoclast activation is the result of several events in the microenvironment without necessarily requiring the existence of a spicific, essential osteoclast activator. On the other hand, a specific promoter of osteoclast differentiation does seem likely to be a product of cells of the stromal/osteoblast series. Evidence in facour of this comes from studies of osteoclast generation in co‐cultures of osteoblast/stromal cells with hemopoietic cells. Conflicting view, maintaining that osteoclasts can develop from hemooietic cells without stromal intervention, might be explaind by varying criteria used in identification of osteoclasts. Osteoblastic and osteoclastic renewal, and the interactions of these lineages, are central to the process of bone remodeling.
p53 Expression in the carcinogenesis in the oral mucosa
Authors: Girod Sabine C. , Krämer Christoph , Knüfermann Ralf , Krueger Garhard R. F.
Published: May 2005

Hyperplastic lesions of the oral mucosa such as leukoplakia and oral lichan planus can eventually develop into squamous cell carcinomas. In the clinical treatment of these lesions it would be very important tobe able to predict the biological behaviour of an individual lesion. In 64 hyperplastic lesions and 85 squamous cell carcinomas of the oral mucosa, the expression of the mutant tumor suppressor gene p53and the grade of dysplasia of the lesions.
V‐sis induces Egr‐1 expression by a pathway mediated by c‐Ha‐ras
Authors: Huang Ruo‐Pan , Ngo Lan , Okamura Daryl , Tucker Mark , Adamson Eileen D.
Published: May 2005

The early growth response gene, Egr‐1, is up‐regulated transiently by mitogens and many other simuli in all cells tested. Using NIH3T3 cells conditionally expressing v‐sis from a metallothionein promoter, we show that the addition of Zn2+ stimulates the production of PDGF‐B(v‐sis) and elicits the expression of Egr‐1 in a dose‐dependent and time‐regulated manner. The signal is likely independent of protein kinase C, but depends on tyrosine kinase and other kinase activities and is mediated by c‐Ha‐Ras since the presence of dominant‐negative mutants of Ras and Raf abrogates the induction of Egr‐1 expression by Zn2+. Transiently activated Ras expression in NIH3T3 cells also stimulates the transient expression of Egr‐1, but cells that constitutively express Rass do not have elevated levels of Egr‐1. Transient assays also demonstrated that Zn2+ or activated Ras expression stimulated the activity of a 950 bp Egr‐1 promoter‐reporter gene construct and this is abrogated in the presence of mutant Ras and Raf. The accumulated data show that Egr‐1 gene expression is regulated by multiple mechanisms, as would be needed for putative role in Cell proliferation, in suppression of transformation and in differentiation.
Analysis of regulatory regions in the COL1A1 gene responsible for 1,25‐dihydroxyvitamin D3‐mediated transcriptional repression in osteoblastic cells
Authors: Pavlin Dubravko , Bedalov Antonio , Kronenberg Mark S. , Kream Barbara E. , Rowe David W. , Smith Catharine L. , Pike J. Wesley , Lichtler Alexander C.
Published: May 2005

The synthesis of type 1 colagen in bone cells is inhibited by the calcium‐regulating hormone 1,25‐dihydroxyvitamin D3. Earlier work from our laboratoties has indicated that vitamin D regulation is at the level of transcription, based on result from both nuclear run‐off assays and functional analysis of a hybrid gene consisting of a 3.6 kb COL1A1 promoter fragment fused to the chloraphenicol acetyltransferase reporter gene. In the present study, we investigated the molecular basis for vitamin D‐mediated transcriptional repression of the COL1A1 gene and report the identification of a region within the COL1A1 upstream promoter (the Hindlll‐Pstl restriction fragment between nucleotides‐2295 and ‐1670) which is necessary for 1,25‐dihydroxyvitamin D3 responsiveness in osteoblastic cells. This hormone‐mediated inhibitory effect on the marker gene parallels the inhibition of the endogenous collagen gene. A 41 bp fragment from this region (between nucleotides‐2256 and ‐2216) contains a sequence which is very similar to vitamin D‐responsive elements identified in the osteocalcin gene. Estracts that binds specifically to this 41 bp fragment, as demonstrated by bandshift anslysis. However, deletion of this vitamin D receptor binding region from either a‐3.5 kb or a‐2.3 kb promoter fragment did not abolish vitamin D responsiveness. These results indicate that a vitamin D response element similar to that described for other D responsive genes (osteocalcin and osteopontin) does not alone mediate the repression of COL1A1 by 1,25‐dihydroxyvitamin D3.
Cultured AIDS‐related Kaposi's sarcoma cells retain a proliferative bioenergetic profile but demonstrate reduced cytoprotective capabilities
Authors: Mallery S. R. , Ng‐Bautista C. L. , Lantry L. E. , Ness G. M. , Hegtvedt A. K. , Lazo A. , Bailer R. T. , Hout B. L. , Stephens R. E. , Brierley G. P.
Published: May 2005

Features of AIDS‐related Kaposi's sarcoma (AIDS‐KS), such as the multifocal presentation at mucosal and epidermal sites subjected to trauma, suggest that AIDS‐KS is initially a reactive hyperplasia that subsequently progresses to a neoplasia. It is recognized that there is an association between sustained states and the subsequent development of neoplasia (e.g., ulcerative colitis/colonic adenocarcinoma). Furthermore, patients who develop AIDS‐KS experience both a constant immune stimulation due to sustained high levelsof virus‐induced cytokines and, because of a sparing effect on their phagoctic cells, retention of the phagocytic inflammatory response. A component of phygocytic activation is the initiation of the oxidative brust, resulting in the generation of reactive oxygen species (ROS), which can be mutagenic to host cells if released beyond the phagolysosome and not inactivated. Our results demonstrate that cultured AIDS‐KS cells possess drcreased cytoprotective capabilities. Relativeto either dermal fibroblasts, or human microvascular endothelial cells (HMECs), AIDS‐KS cells contained significantly lower levels of glutathione, a tripeptide integral in both cytoprotection and maintenance of cellular thiol status. While HMECs increased catalase activity during culture in the cytokine‐rich KS milieu (control medium supplement with conditioned medium from MOT, an TLV II‐infected cell line), AIDS‐KS cells demonstrated reduced catalase function under these conditions. Furthermore, HMEC cultures showed in inherent biochemical responsiveness, by increasing catalase activity following exposure to exaogenous H2(O2). In contrast, the catalase activity of AIDS‐KS cells decreased following (H2O2) challenge. Our results show that an inherent deficiency in cellular cytoprotection is present in AIDS‐KS cells and suggest that oxidant stress may function in the development and progression AIDS‐KS.