Development of "heparin-like" polymers using swift heavy ion and gamma radiation. I. Preparation and characterization of the materials.

The development of ideal antithrombogenic polymers, a major problem in biomaterials sciences, is a primary objective in the fields of cardiovascular prostheses, artificial hearts, and other devices. To decrease their thrombogenicity, which remains the major obstacle, we have developed polymeric materials endowed with a specific affinity for antithrombin III (ATIII) and thus able, like heparin, to catalyze the inhibition of thrombin by ATIII. Sulfonate and sulfonamide groups are introduced onto phenyl rings belonging to styrene residues, which are radiation grafted (using swift heavy ion and gamma radiation) onto poly(vinylidene difluoride) (PVDF) and also onto poly(vinylidene fluoride/hexafluoropropylene) [P(VDF-HFP)]. In contrast to gamma radiation, which leads to a homogeneous modification, the advantage of swift heavy ion grafting is that only small regions are modified; thus, the surface may present hydrophilic (corresponding to the modified areas) and hydrophobic microdomains (corresponding to the unmodified areas) of different sizes, depending on the absorbed dose and grafting yield. Surface topography and composition are characterized by scanning electron microscopy (SEM) and atomic force microscopy (AFM). Sulfur, sodium, fluorine, and carbon are determined by scanning electron microscopy combined with energy-dispersive X-ray analysis (SEM-EDXA). The amount of fluorine decreases as polystyrene (PS) is grafted, whatever the kind of radiation and polymer. When the polymers are functionalized, the amount of fluorine also decreases while sodium and sulfur appear. Functionalization seems to increase the roughness of the surface, and its area.

The c-myc coding region determinant-binding protein: a member of a family of KH domain RNA-binding proteins.

The half-life of c- myc mRNA is regulated when cells change their growth rates or differentiate. Two regions within c- myc mRNA determine its short half-life. One is in the 3'-untranslated region, the other is in the coding region. A cytoplasmic protein, the coding region determinant-binding protein (CRD-BP), binds in vitro to the c- myc coding region instability determinant. We have proposed that the CRD-BP, when bound to the mRNA, shields the mRNA from endonucleolytic attack and thereby prolongs the mRNA half-life. Here we report the cloning and further characterization of the mouse CRD-BP, a 577 amino acid protein containing four hnRNP K-homology domains, two RNP domains, an RGG RNA-binding domain and nuclear import and export signals. The CRD-BP is closely related to the chicken beta-actin zipcode-binding protein and is similar to three other proteins, one of which is overexpressed in some human cancers. Recombinant mouse CRD-BP binds specifically to c- myc CRD RNA in vitro and reacts with antibody against human CRD-BP. Most of the CRD-BP in the cell is cytoplasmic and co-sediments with ribosomal subunits.

Developmental regulation of CRD-BP, an RNA-binding protein that stabilizes c-myc mRNA in vitro.

We previously isolated and characterized a coding region determinant-binding protein (CRD-BP) that might regulate c-myc mRNA post-transcriptionally. CRD-BP binds specifically to the coding region of c-myc mRNA and might stabilize c-myc mRNA in vitro by protecting it from endonucleolytic cleavage. Since c-myc abundance is regulated during embryonic development and cell replication, we investigated whether CRD-BP is also regulated in animal tissues. We focused on CRD-BP expression during rat liver development and liver regeneration, because c-myc mRNA is regulated post-transcriptionally in both cases. CRD-BP expression parallels c-myc expression during liver development; the protein is present in fetal and neonatal liver but is absent or in low abundance in adult liver. In contrast, the up-regulation of c-myc mRNA following partial hepatectomy is not accompanied by up-regulation of CRD-BP. To our knowledge, CRD-BP is the first example of a putative mammalian mRNA-binding protein that is abundant in a fetal tissue but either absent from or scarce in adult tissues. Its expression in fetal liver and in transformed cell lines suggests CRD-BP is an oncofetal protein.

CeReS-18, a novel cell surface sialoglycopeptide, induces cell cycle arrest and apoptosis in a calcium-sensitive manner.

Very few growth inhibitors have been identified which can inhibit the proliferation of a broad spectrum of human breast cancer cell lines. CeReS-18, a novel cell surface sialoglycopeptide growth inhibitor, can reversibly inhibit the proliferation of both estrogen receptor positive (MCF-7) and negative (BT-20) human breast cancer cell lines. In addition, at concentrations above those required for the reversible inhibition of cell proliferation, CeReS-18 can also induce cell death in MCF-7 cells. Changes in nuclear and cytoplasmic morphology, characteristic of apoptosis, were detected in MCF-7 cells treated with a cytotoxic concentration of CeReS-18, and internucleosomal DNA cleavage was also observed. The sensitivity of MCF-7 and BT-20 cells to the biological properties of CeReS-18 could be influenced by altering the calcium concentration in the extracellular growth medium, such that when the calcium concentration in the environment was decreased, and increased sensitivity to CeReS-18-induced growth inhibition and cytotoxicity were observed. The addition of the calcium chelating agent EGTA to MCF-7 cells, cultured in a normal calcium environment, could mimic the increased sensitivity to the biological effects of CeReS-18 observed under reduced calcium conditions.

Inhibition of hormone and growth factor responsive and resistant human breast cancer cells by CeReS-18, a cell regulatory sialoglycopeptide.

We have previously documented that CeReS-18, a cell regulatory sialoglycopeptide, inhibits the cellular proliferation of normal and transformed cell types from a diverse range of species. Most cell types studies exhibit a similar sensitivity to the reversible but growth inhibitory effects of CeReS-18 at 7 x 10(8) M concentration, while at higher concentrations CeReS-18 can elicit cytotoxicity. The present study was conducted to examine the effect of CeReS-18 on the proliferation of human mammary epithelial carcinoma cells. MCF-7 cells, which are estrogen receptor positive (ER+), and BT-20 cells, which are estrogen receptor negative (ER+), were utilized. Both cell lines show equal sensitivity to growth inhibition elicited by CeReS-18. Complete cessation of cell cycling was achieved with 7 x 10(-8) M CeReS-18, and the arrest was shown to be completely reversible. Flow cytometric analysis, performed on CeReS-18 treated cells from both cell types, revealed that the majority of these cells were arrested in the G1 phase of the cell cycle. When cells were treated simultaneously with inhibitor and stimulatory concentrations of mitogens such as epidermal growth factor (EGF), basic fibroblast growth factor (b-FGF), estrogen, insulin-like growth factors I and II (IGFI and IGFII), no alteration of the inhibitory activity of CeReS-18 was observed. CeReS-18 clearly abrogated the mitogenic activity that these growth factors elicited with human mammary carcinoma cells.

Growth inhibition of BALB/c mouse keratinocytes by TGF-beta 1 and CeReS-18 appears to act through distinct mechanisms.

Cell growth is controlled by the complex interactions of both positive and negative growth modulators. Studies were performed to directly compare the growth inhibitory properties of TGF-beta 1 and CeReS-18, a novel cell surface sialoglycopeptide growth inhibitor. Growth inhibition by CeReS-18 and that by TGF-beta 1 shared some similarities although significant differences were apparent. Similarities included a dose-responsive inhibition of BALB/c mouse keratinocyte (MK) cell proliferation that could be nontoxic and reversible. Both CeReS-18 and TGF-beta 1 could equally inhibit the stimulation of DNA synthesis induced by serum or keratinocyte growth factor in MK cells, and in both cases the inhibition was not due to decreased KGF binding to the target cell surface receptor. Inhibition of cell proliferation with CeReS-18, followed by an immediate reincubation with either CeReS-18 or TGF-beta 1, suggested that the sites of arrest mediated by both inhibitors were similar but not necessarily identical. However, recovery from CeReS-18-induced growth inhibition could be achieved by either removing the CeReS-18 or adding calcium directly to CeReS-18-inhibited MK cells, while recovery from TGF-beta 1-induced growth arrest could be reversed only by TGF-beta 1 removal. In addition, the sensitivity of MK cells to CeReS-18-induced cell cycle arrest could be altered by changing the extracellular calcium concentration, but sensitivity to TGF-beta 1 was unaffected by the calcium environment of the MK cells. While recovery from cell cycle arrest was rapid and complete with MK cell cultures inhibited with CeReS-18, removal of TGF-beta 1 led to a slower and incomplete recovery of cell cycling.

Role of calcium in growth inhibition induced by a novel cell surface sialoglycopeptide.

Our laboratory has purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) from intact bovine cerebral cortex cells. Evidence presented here demonstrates that sensitivity to CeReS-18-induced growth inhibition in BALB-c 3T3 cells is influenced by calcium, such that a decrease in the calcium concentration in the growth medium results in an increase in sensitivity to CeReS-18. Calcium did not alter CeReS-18 binding to its cell surface receptor and CeReS-18 does not bind calcium directly. Addition of calcium, but not magnesium, to CeReS-18-inhibited 3T3 cells results in reentry into the cell cycle. A greater than 3-hour exposure to increased calcium is required for escape from CeReS-18-induced growth inhibition. The calcium ionophore ionomycin could partially mimic the effect of increasing extracellular calcium, but thapsigargin was ineffective in inducing escape from growth inhibition. Increasing extracellular calcium 10-fold resulted in an approximately 7-fold increase in total cell-associated 45Ca+2, while free intracellular calcium only increased approximately 30%. However, addition of CeReS-18 did not affect total cell-associated calcium or the increase in total cell-associated calcium observed with an increase in extracellular calcium. Serum addition induced mobilization of intracellular calcium and influx across the plasma membrane in 3T3 cells, and pretreatment of 3T3 cells with CeReS-18 appeared to inhibit these calcium mobilization events. These results suggest that a calcium-sensitive step exists in the recovery from CeReS-18-induced growth inhibition. CeReS-18 may inhibit cell proliferation through a novel mechanism involving altering the intracellular calcium mobilization/regulation necessary for cell cycle progression.

Ha-ras p21-GTP levels remain constant during primary keratinocyte differentiation.

Several lines of evidence that indicate that mutation of the Ha-ras oncogene is the initiating event in mouse skin carcinogenesis. Keratinocytes known to possess a mutated Ha-ras have been shown to be resistant to differentiation. Thus, overstimulation of the Ha-ras signaling pathway appears to block normal keratinocyte differentiation, and we hypothesized that for normal keratinocytes to terminally differentiate, the Ha-ras signaling cascade must be turned off. In the present studies, we measured the level and activity state of Ha-ras p21 protein in cultured keratinocytes undergoing calcium-induced differentiation. We have employed Western blot analysis to demonstrate that Ha-ras p21 protein levels remain constant during primary newborn and adult keratinocyte differentiation. The overall level of Ha-ras p21 was higher in immortalized, benign, and malignant mouse keratinocyte cell lines than in normal keratinocytes but did not change within each cell type when subjected to differentiating conditions. The percentage of Ha-ras p21 protein in its active, GTP-bound form also remained unchanged during primary adult keratinocyte differentiation and in immortalized, benign, and malignant keratinocytes subjected to differentiating conditions. Our results indicate that terminal differentiation of primary adult mouse keratinocytes occurred in the presence of constant levels of Ha-ras p21-GTP, suggesting that the Ha-ras signaling pathway may be blocked at a point distal to a step involving the Ha-ras p21 protein itself.

Calcium influences sensitivity to growth inhibition induced by a cell surface sialoglycopeptide.

While studies concerning mitogenic factors have been an important area of research for many years, much less is understood about the mechanisms of action of cell surface growth inhibitors. We have purified an 18 kDa cell surface sialoglycopeptide growth inhibitor (CeReS-18) which can reversibly inhibit the proliferation of diverse cell types. The studies discussed in this article show that three mouse keratinocyte cell lines exhibit sixty-fold greater sensitivity than other fibroblasts and epithelial-like cells to CeReS-18-induced growth inhibition. Growth inhibition induced by CeReS-18 treatment is a reversible process, and the three mouse keratinocyte cell lines exhibited either single or multiple cell cycle arrest points, although a predominantly G0/G1 cell cycle arrest point was exhibited in Swiss 3T3 fibroblasts. The sensitivity of the mouse keratinocyte cell lines to CeReS-18-induced growth inhibition was not affected by the degree of tumorigenic progression in the cell lines and was not due to differences in CeReS-18 binding affinity or number of cell surface receptors per cell. However, the sensitivity of both murine fibroblasts and keratinocytes could be altered by changing the extracellular calcium concentration, such that increased extracellular calcium concentrations resulted in decreased sensitivity to CeReS-18-induced proliferation inhibition. Thus the increased sensitivity of the murine keratinocyte cell lines to CeReS-18 could be ascribed to the low calcium concentration used in their propagation. Studies are currently under way investigating the role of calcium in CeReS-18-induced growth arrest. The CeReS-18 may serve as a very useful tool to study negative growth control and the signal transduction events associated with cell cycling.

DNA-mediated gene transfection into primary cultures of adult mouse keratinocytes.

An efficient and reproducible technique for the transfection of primary cultures of adult mouse keratinocytes has been developed. The procedure involves culturing the primary adult mouse epidermal cells at 32 degrees C in an enriched media until they reach 70 to 95% confluency, followed by transfection with exogenous DNA in a low potassium environment. Using chloramphenicol acetyl transferase (CAT) transient gene expression assays and various strong viral promoter/CAT constructs, the transfection procedure was optimized for media formulation, plasmid DNA concentration, carrier DNA concentration, incubation temperature, incubation period, and cell density. Optimized parameters include the use of 6 micrograms plasmid DNA and 10 micrograms pUC19 carrier DNA per 60-mm tissue culture dish. Since primary keratinocytes undergo a well-characterized pattern of differentiation in vitro in response to extracellular calcium concentrations, this transfection procedure should provide a useful model in which to study both tissue- and differentiation-specific gene expression.

Resistance of adult keratinocytes to differentiation-induced decrease in Ha-ras mRNA levels observed in newborn keratinocytes.

During two-stage mouse skin tumorigenesis, the mouse c-Ha-ras oncogene undergoes activation by point mutation after initiation with polycyclic aromatic hydrocarbons. Furthermore, initiated epidermal cells containing an activated Ha-ras oncogene have been shown to be resistant to calcium-induced terminal differentiation. However, the relationship between Ha-ras expression and the differentiation process is not well understood in either normal or initiated cells. Before attempting to explore the role of Ha-ras expression in epidermal differentiation during tumorigenesis, we felt that investigation of Ha-ras gene expression in normal primary epidermal cells undergoing differentiation was warranted, since primary cultures of normal newborn and adult keratinocytes presumably contain the stem cells from which skin tumors arise. In the present studies, northern blot analysis was used to compare Ha-ras expression in normal newborn and adult epidermal cells undergoing differentiation. Steady-state levels of Ha-ras mRNA remained unchanged in primary cultures of normal adult epidermal cells during calcium-induced differentiation, whereas steady-state levels of Ha-ras transcripts decreased during calcium-induced differentiation in primary newborn epidermal cells. Differentiation was induced by switching the adult and newborn keratinocytes from medium containing 0.05 mM Ca2+ to medium containing one of three different calcium concentrations (0.15, 0.5, or 1.2 mM Ca2+). The decrease in Ha-ras mRNA levels observed during differentiation in newborn keratinocytes occurred as an intermediate event in the differentiation process, was specific for the Ha-ras gene, and was not due to a general decrease in transcriptional activity during differentiation. Characteristic patterns of keratin 14 gene expression and cornified envelope formation were observed, verifying that the differentiation process had been induced in both the primary adult and newborn epidermal cells. That adult keratinocytes are resistant to the differentiation-induced reduction in Ha-ras mRNA expression observed in newborn keratinocytes may explain the difference in in vivo tumorigenic potentials of newborn and adult skin.

Transient expression of the cloned mouse c-Ha-ras 5' upstream region in transfected primary SENCAR mouse keratinocytes demonstrates its power as a promoter element.

The mouse Ha-ras oncogene is activated by point mutation and overexpressed in developing papillomas during two-stage skin carcinogenesis in SENCAR mice. One of our research aims is to characterize the factors regulating Ha-ras gene expression at the transcriptional level in SENCAR mouse epidermis. Towards this goal, we sequenced 1400 bp of the 5' upstream region of the mouse Ha-ras gene so as to characterize various cis-regulatory elements present in the gene. We identified seven sites with the proper consensus sequence for binding the SP1 transcription factor and three potential binding sites for the CTF-1 factor. In addition, we located a 13-base sequence with 92% homology to the consensus sequence for an estrogen response element and two hexamers with consensus sequences identical to the core sequence of the glucocorticoid response element. A series of transient gene expression vectors was constructed in which various regions of the mouse Ha-ras 5' upstream region were fused to the chloramphenicol acetyltransferase (CAT) gene. These expression plasmids were transfected into newborn and adult primary SENCAR epidermal cells, the epidermal cell population that presumably contains the stem cells involved in two-stage skin tumorigenesis. Transient gene expression assays carried out after 48-72 h indicated that a 2.3-kb Ha-ras 5' fragment produced CAT activity comparable to that produced by pSV2CAT and pdolCMVCAT, both of which are plasmids with strong viral promoters and enhancers driving CAT gene expression. Maintenance of transfected keratinocytes under both nondifferentiating (0.05 mM calcium) and differentiating (1.2 mM calcium) culture conditions demonstrated that the mouse Ha-ras upstream region was relatively unresponsive to changes in calcium concentration in transient expression assays carried out in either newborn or adult keratinocytes. Our results demonstrated the power of the cloned mouse Ha-ras promoter and upstream region in driving transient gene expression after transfection into primary keratinocytes.

Binding of the 97 kDa glucocorticoid receptor to the 5' upstream flanking region of the mouse c-Ha-ras oncogene.

Glucocorticoids regulate transcription of specific genes through the interaction of glucocorticoid hormone receptor complexes with DNA binding sites called glucocorticoid response elements (GREs). The GRE consensus sequence has been defined to be the imperfect palindromic sequence 5'-GGTACANNNTGTTCT-3', the most highly conserved portion being the 5'-TGTTCT-3' hexamer. We have identified 5 potential GREs in the 5' upstream noncoding region of the mouse c-Ha-ras oncogene, two with the same hexanucleotide sequence and three with a similar sequence. When subcloned fragments of the mouse c-Ha-ras 5' upstream region (containing the 2 hexamer GREs of exact homology) were fused to the chloramphenicol acetyltransferase (CAT) reporter gene and transfected into HeLa cells, CAT expression driven from the ras promoter was induced up to 3-fold in the presence of dexamethasone. To determine whether the 5' upstream region of the mouse Ha-ras gene was capable of specifically interacting with the glucocorticoid receptor complex, we performed Southwestern blot analysis showing that cloned DNA fragments from the 5' upstream region of the mouse c-Ha-ras gene were able to bind a 97 kDa protein in whole cell extracts from both primary SENCAR mouse epidermal cells and HeLa G cells. Immunodepletion of the epidermal cell extract with a monoclonal antibody to the glucocorticoid receptor verified that the 97 kDa protein bound by the Ha-ras 5' region was indeed the glucocorticoid receptor protein. Our results demonstrate that the upstream noncoding region of the mouse c-Ha-ras gene binds the glucocorticoid receptor. Furthermore, the presence of glucocorticoids enhances the transcription of the mouse Ha-ras promoter region in a transient gene expression assay.

Factors influencing intake of high urea-containing rations by lactating dairy cows.

In a series of experiments we investigated effects of several factors on intake of urea by lactating dairy cows. Cows given an unfamiliar ingredient or altered moisture in the ration reduced their intake, and this effect was attributed to a newness factor. Addition of urea to a ration may have a similar effect. An experimental design providing both no-choice and two-choice data was devised to minimize the effect of newness. When urea was isolated physically by pellets coated with ground corn, cows selected against urea-containing pellets on a two-choice basis and reduced intake on a no-choice basis. When the choice was between two urea-containing rations, cows preferred the pellets in which urea flavor and odor should have been most evident. Urea odor in the feedbox or urea in the drinking water did not reduce feed intake. Eating rate of a 2.5% urea-containing ration over two daily 30-min feeding periods was about one-half that of a non-urea ration. This effect was evident during the first 5-min eating interval. Administration of urea solution into the rumen prior to feeding a nonurea ration markedly reduced subsequent intake. Reticulum pH and ammonia indicated possible sublethal ammonia toxicity. Intake and eating rate were depressed and reticulum pH was elevated when cows were fed a ration with 2.5% urea versus 1% urea or nonurea rations. Elevated reticulum ammonia occurred on both 1 and 2.5% urea-containing rations. Cows not previously exposed to urea-containing rations reduced intake and eating rate when fed rations with 2.0 and 2.5% urea. Intake reduction was not comparable to that by cows preconditioned to urea rations. Upon third exposure to 2.5% urea in the ration, cows reduced and ceased intake but readily consumed a nonurea ration. Cows require preconditioning to develop a negative aversion to rations containing high urea, perhaps through a mechanism of sublethal ammonia toxicity.