Effect of cytostatic agents on expression levels of human beta-defensins-1-4 in A431 and MCF-7 cell lines.

The aim of the study was to analyze an effect of cytostatic agents of different mechanism of action on expression levels of human beta-defensins-1-4 (hBD-1-4) in cultured human cancer cell lines. MATERIALS AND METHODS: Expression levels of hBD-1-4 mRNA were assessed using qPCR in human epidermoid carcinoma A431 cells and human breast adenocarcinoma MCF7 cells treated with cisplatin, methotrexate, doxorubicin or vincristine at the IC20 concentrations. RESULTS: The cytostatic agents with different mechanisms of action affected differently expression of hBDs, dependent on the cell line. Mostly, cytostatic agents suppressed significantly expression of hBDs. In contrast, vincristine caused significant up-regulation of hBD-1 (12 fold, p < 0.05) and hBD-4 (2 fold, p < 0.05) in MCF7, and doxorubicin significantly enhanced expression of hBD-3 (2 fold, p < 0.05) and hBD-4 (> 10 fold, p < 0.05) in A431 cells. CONCLUSION: The results of this pilot study show that expression levels of hBD-1-4 may be altered upon treatment with cytostatic agents depending on nature of cells.

Resistance of Arctic phytoplankton to ocean acidification and enhanced irradiance.

The Arctic Ocean is a region particularly prone to ongoing ocean acidification (OA) and climate-driven changes. The influence of these changes on Arctic phytoplankton assemblages, however, remains poorly understood. In order to understand how OA and enhanced irradiances (e.g., resulting from sea-ice retreat) will alter the species composition, primary production, and eco-physiology of Arctic phytoplankton, we conducted an incubation experiment with an assemblage from Baffin Bay (71°N, 68°W) under different carbonate chemistry and irradiance regimes. Seawater was collected from just below the deep Chl a maximum, and the resident phytoplankton were exposed to 380 and 1000 µatm pCO2 at both 15 and 35% incident irradiance. On-deck incubations, in which temperatures were 6 °C above in situ conditions, were monitored for phytoplankton growth, biomass stoichiometry, net primary production, photo-physiology, and taxonomic composition. During the 8-day experiment, taxonomic diversity decreased and the diatom Chaetoceros socialis became increasingly dominant irrespective of light or CO2 levels. We found no statistically significant effects from either higher CO2 or light on physiological properties of phytoplankton during the experiment. We did, however, observe an initial 2-day stress response in all treatments, and slight photo-physiological responses to higher CO2 and light during the first five days of the incubation. Our results thus indicate high resistance of Arctic phytoplankton to OA and enhanced irradiance levels, challenging the commonly predicted stimulatory effects of enhanced CO2 and light availability for primary production.

Angiotensin II signal transduction pathways.

It has been 100 years since the discovery of renin by Tigerstedt and Bergman. Since that time, numerous discoveries have advanced our understanding of the renin-angiotensin system, including the observation that angiotensin II is the effector molecule of this system. A remarkable aspect of angiotensin II is the many different physiological responses this simple peptide induces in different cell types. Here, we focus on the signal transduction pathways that are activated as a consequence of angiotensin II binding to the AT1 receptor. Classical signaling pathways such as the activation of heterotrimeric G proteins by the AT1 receptor are discussed. In addition, recent work examining the role of tyrosine phosphorylation in angiotensin II-mediated signal transduction is also examined. Understanding how these distinct signaling pathways transduce signals from the cell surface will advance our understanding of how such a simple molecule elicits such a wide variety of specific cellular responses.

Expression of testis angiotensin-converting enzyme is mediated by a cyclic AMP responsive element.

Testis angiotensin-converting enzyme (testis ACE), an ACE isozyme that plays an important role in male fertility, is transcribed from a unique promotor active only in developing spermatids. In vitro analysis suggests the importance of a cyclic AMP response element (CRE)-like region within the testis ACE promoter, and similar DNA motifs are important in the expression of a variety of testis-specific genes. In the present study, we examined the effects of mutations in the CRE-like element on testis ACE promoter activity in vivo using transgenic mice. Disruption of this element reduced reporter gene expression to near background levels. In contrast, conversion of the CRE-like element to a consensus CRE-binding site resulted in high level expression of the reporter gene specifically in the testis. These experiments prove that the CRE-like element is essential for testis ACE promoter activity, although it does not appear to be responsible for its tissue specificity. These data provide insight into how a phenotypically differentiated tissue, ie, male gem cells, regulate tissue-specific gene expression.

Similarity between natural and recombinant human alpha-fetoprotein as inhibitors of estrogen-dependent breast cancer growth.

Alpha-fetoprotein (AFP) isolated from rodent amniotic fluid or human cord sera, upon incubation with a molar excess of estradiol, is converted to a form which inhibits estrogen-stimulated tissue growth. The purpose of this study was to determine whether recombinant human AFP produced in an E. coli expression system retained this function. The recombinant protein was similar to the natural protein isolated from pooled human cord sera in all functional aspects evaluated. It was detected by monoclonal and polyclonal antibodies to the natural protein. Following exposure to estradiol, it was converted to an inhibitor of estrogen-stimulated growth of immature mouse uterus yielding a dose/response curve similar to that of the natural protein. It inhibited the growth of estrogen-dependent (MCF-7) but not estrogen-independent (MDA-MB-231) breast cancer xenografts with the same schedule dependency and resultant histological changes as the natural protein. Availability of large quantities of homogeneous, biologically active recombinant human AFP will facilitate further studies of structure/function, mechanism, and therapeutic potential of this agent as a regular of breast cancer growth.

Purification and characterization of human and mouse recombinant alpha-fetoproteins expressed in Escherichia coli.

Alpha-fetoprotein (AFP) is a tumor-associated embryonic molecule whose precise biological function(s) remains unclear. A more complete analysis of the physiological activities of this oncofetal protein has, until now, been severely limited by the lack of an appropriate source from which to obtain pure AFP in any sizeable quantity. In the present investigation, we obviate this problem by cloning and efficiently overexpressing mature mouse and human AFP cDNA's in Escherichia coli. For recombinant mouse AFP (rMoAFP), large segments of the coding region were excised from the preexisting plasmids pAFP1 and pAFP2, which together encompass 90% of the AFP sequence. The mouse cDNA was made complete by the addition of N- and C-terminal encoding oligonucleotides. Mouse AFP cDNA was expressed directly as a full-length molecule in vector pTrp4 or as fusion proteins in plasmids pMALc and pRX1 under the transcriptional control of trp or tac promoters. Accumulation of rMoAFP was significantly increased in protease-deficient E. coli strains over nonprotease-deficient strains, > or = 10% of total cell protein. Of the gene fusion proteins examined, none offered significant advantage over the direct expression product in terms of recombinant protein stability, overall levels of synthesis, or facilitated purification. Recombinant AFP polypeptides expressed by pTrp4 were as expected, deposited in bacterial inclusion bodies. Subsequent to resolubilization/refolding, rMoAFP was first enriched by passage over Q-Sepharose resin followed by final purification using immobilized copper-chelate affinity chromatography. Protein sequencing of the N-terminus revealed that purified rMoAFP had a deletion of the first nine amino acids coded for by the full-length mouse AFP cDNA. Similar N-terminal deletions are observed with AFP isolates originating from natural sources. A complete human AFP cDNA was generated from a fetal liver cDNA library and was cloned into vector pTrp4. Recombinant human AFP (rHuAFP) was expressed under the identical conditions employed for rMoAFP but purification had to be modified to include preparative Mono Q anion exchange chromatography. N-terminal sequencing, amino acid compositional analysis, and electrospray mass spectrometry revealed that purified rHuAFP was intact and unaltered and that the initiator methionine was completely removed. The biological activity of recombinant AFP, as judged by its inhibitory effects on in vitro lymphocyte proliferation, was equivalent to that of the native protein. The availability of large quantities of mouse and human recombinant AFP molecules should now permit detailed structure-function analyses of this important oncofetal protein to proceed in a manner unimpeded by previous limitations in both quantity and quality of the native proteins.

Evidence that immunosuppression is an intrinsic property of the alpha-fetoprotein molecule.

Among the proteins that comprise the albumin family, alpha-fetoprotein (AFP) is the only member which exhibits immunoregulatory properties. However, some investigations have argued that AFP-mediated immunosuppression is not an inherent property of the molecule itself, but is instead, hypothesized to be either a function of a low molecular weight inhibitor bound to AFP or to a post-translational modification of the protein. AFP cannot be isolated from natural sources in quantities sufficient for the detailed biochemical and functional analyses required to resolve these issues. We have therefore produced recombinant forms of the protein (rAFP) by cloning the cDNA's for mouse and human AFP in both eukaryotic and prokaryotic expression systems. As described in this report, we were able to abundantly express rAFP's in bacterial, baculovirus and yeast expression systems. Recombinant proteins derived from each expression system were recognized by polyclonal and monoclonal anti-AFP antibodies as determined by immunoblot analysis. Pure recombinant protein samples, as characterized by polyacrylamide gel analyses, N-terminal sequencing and FPLC and HPLC chromatography, were evaluated for their immunoregulatory properties in murine and human in vitro immunological assays. The results of these studies establish that rAFP is functionally equivalent to natural fetal derived AFP molecules. Importantly, the data reported here demonstrate that AFP-mediated immunoregulation is an activity intrinsic to the molecule itself and cannot be attributed to either putative non-covalently bound moieties or to post-translational modifications such as glycosylation and sialylation. These studies provide a basis for initiating detailed investigations into the potential clinical usefulness of AFP as an immunotherapeutic agent.