Collagen sandwich culture affects intracellular polyamine levels of human hepatocytes

Abstract. Extracellular matrices, like collagen layers, play an important role in preventing dedifferentiation of hepatocytes in long-term culture experiments. It has also been shown that polyamines are crucial for cell growth and liver differentiation – regeneration. Primary cultured hepatocytes with their low mitotic activity might be a valuable tool in studying the role of polyamines in differentiation. Here, our goal was to investigate whether an extracellular cell culture matrix can influence intracellular polyamine levels in human hepatocytes during long-term culture. Primary human hepatocytes were isolated from surgical tissue resections and were maintained either in single collagen (SG) or double collagen gel (DG) layer (sandwich) culture systems. Cell viability and function were examined and intracellular polyamine levels were measured using a highly sensitive high performance liquid chromatography (HPLC) method. Hepatocytes showed high viability in both culture systems used, but albumin secretion was diminished in SG cultured hepatocytes after 14 days. In general, total intracellular polyamine levels of hepatocytes decreased markedly in both SG and DG within the first days of culture, but remained constant until day 21 with a SG/DG ratio of about 1.4. Individual polyamines levels were dependent on the culture time and system, where spermine decreased and putrescine increased in both SG and DG over time (day 14), but spermidine increased only in DG. Our results suggest that polyamine levels, in particular putrescine, might be important regulators of hepatocyte specific function in vitro and therefore serve as a marker of differentiation for cultivated human hepatocytes.     

Control of Leishmania mexicana proliferation by modulation of polyamine intracellular levels

Repeated treatments of Leishmania mexicana promastigote cultures with a-difluoromethylornithine could not block proliferation when the parasite was grown in a rich medium. Although the irreversible inhibitor of ornithine decarboxylase was able to abolish the enzymatic activity under these conditions, polyamine depletion was only partial probably due to the uptake of these substances from the external medium. Conversely, when Leishmania was cultivated in a defined medium essentially free of polyamines, a-difluoromethylornithine was able to decrease the growth rate and proliferation was arrested after several passages in the presence of the drug. Parasite multiplication could be resumed by addition of exogenous polyamines, and a strict correlation between Leishmania promastigote growth and intracellular levels of spermidine was observed.     

Promoters maintain their relative activity levels under different growth conditions

Most genes change expression levels across conditions, but it is unclear which of these changes represents specific regulation and what determines their quantitative degree. Here, we accurately measured activities of ～900 S. cerevisiae and ～1800 E. coli promoters using fluorescent reporters. We show that in both organisms 60–90% of promoters change their expression between conditions by a constant global scaling factor that depends only on the conditions and not on the promoter's identity. Quantifying such global effects allows precise characterization of specific regulation—promoters deviating from the global scale line. These are organized into few functionally related groups that also adhere to scale lines and preserve their relative activities across conditions. Thus, only several scaling factors suffice to accurately describe genome‐wide expression profiles across conditions. We present a parameter‐free passive resource allocation model that quantitatively accounts for the global scaling factors. It suggests that many changes in expression across conditions result from global effects and not specific regulation, and provides means for quantitative interpretation of expression profiles.     

Epidermal proteins of cultured human and bovine keratinocytes.

Bovine and human epidermal cells were cultured on mitomycin C treated fibroblasts. The cells were carried through four passages and found to synthesize fibrous proteins and insoluble cell envelopes. Acid buffer soluble fibrous protein, prekeratin, and urea soluble fibrous protein were both identified and the latter was the major component in older cultures. Some of the prekeratin polypeptides of intact tissue were not found in cultured cells, but the ones that were present corresponded to those of whole tissue. X-ray diffraction, amino acid analysis and immunological techniques were used to establish that the polypeptides were keratins. The insoluble cell envelopes had a higher proline and 1/2 cystine content than the fibrous protein, similar to what is found in whole epidermis. Histidase, a characteristic enzyme marker of whole epidermis, was not observed in cultured cells. These studies indicate that differentiation occurs in cultured cells but it may not be as complete as in intact tissue.     

Human keratinocytes maintain reversible anti-apoptotic defenses in vivo and in vitro

Human keratinocytes proliferate and differentiate in an epidermal environment where induction of apoptosis can be triggered by ultraviolet radiation (UVR), activated lymphocytes and cytokines. The purpose of this study was to determine whether keratinocytes were susceptible to apoptosis induced by ionophore, ultra-violet radiation, cytokines or crosslinking of CD95 (Fas/APO-1). In normal human skin exposed to two minimal erythema doses of ultraviolet radiation, suprabasal cells were the first keratinocytes to demonstrate apoptotic nuclei, and by 48 h apoptotic cells were identified throughout the mid to upper epidermis. However, most keratinocytes resisted apoptosis and UVR-induced apoptosis was not observed in basal cells, or in the most differentiated epidermis. Human keratinocytes and keratinocyte cell lines cultured in vitro developed maximal apoptosis 48 h after radiation. Human keratinocytes cultured in full growth factor supplements were resistant to UVR-induced apoptosis compared to keratinocyte cell lines or to a lymphoid cell line (HL60) susceptible to apoptosis. Keratinocyte cell lines were completely resistant to apoptosis induced by interferon-, interferon-, IL-2, IL-6, TNF-, IL-1Ra, and GM-CSF. A subset of the cells in cultures of keratinocytes and transformed keratinocyte cell lines died by apoptosis in response to anti-Fas, IL-1 and TNF- plus IFN- and ionophore. Second passage freshly isolated human keratinocytes were much more resistant to apoptosis induced by ionophore, anti-Fas and cytokines than were transformed keratinocyte cell lines. Calcium shift to induce differentiation in second-passage keratinocyte cultures made keratino-cytes even more resistant to UVR-induced apoptosis. This parallels the lack of UVR-induced apoptosis observed in the most differentiated keratinocytes in irradiated human skin. Both keratinocytes and kerati-nocyte cell lines express rather low levels of the anti-apoptotic proteins bcl-2 and bcl-x compared to other apoptosis-resistant cell types. The differences between keratinocytes and keratinocyte cell lines in suscepti-bility to apoptosis are not explained by difference in expression of bcl-2 or bcl-x. Finally, withdrawal of growth factors from keratinocytes decreased cell survival following UVR and increased the induction of apoptosis. Inhibition of protein synthesis with cyclo-heximide also made keratinocytes more susceptible to UVR-induced apoptosis, indicating that anti-apop-totic defences in cultured keratinocytes are dependent on active protein synthesis. These experiments show that the strong keratinocyte defences against apoptosis are stratified within the epidermis, and can be altered by differentiation and growth factor withdrawal.     

Features of the intracellular repair of irradiated keratinocytes

By means of light and electron microscopy, intracellular reparation has been studied after a local x-ray radiation of the rat paws (7.74 X 10(-1) Ci/kg) using radioprotectors (mexamin, cysteamin, ionol) and other chemical compounds (including membranoprotective ones). Restoration of the intracellular structures after x-ray burns proceeds more slowly and more complexly than reparation of the epidermis as a tissue system. To the slowly repairing intracellular formations belong mitochondria and, especially, internal mitochondrial membrane, as well as intercellular contacts. Under radiation mitochondria increase their volume at the expense of their three-fold swelling. Preliminary treatment of the skin with some of the compounds mentioned above decreases or completely prevents these changes. By means of the membranoactive chemical compounds, as well as by means of the known radioprotectors it is possible essentially to normalize the process of intracellular reparation and physiological regeneration of the ultrastructures, and in some cases, to stimulate reparative processes in them.     

Epidermal growth factor: modulator of murine embryonic palate mesenchymal cell proliferation, polyamine biosynthesis, and polyamine transport

Polyamines (putrescine, spermidine, and spermine) are normal cellular constituents able to modulate cellular proliferation and differentiation in a number of tissues and cell types. This investigation explores the response of murine embryonic palate mesenchymal (MEPM) cells to epidermal growth factor (EGF) in terms of biosynthesis of putrescine and its transport across the plasma membrane and tests the hypothesis that polyamine transport can serve as an alternative mechanism (other than biosynthesis) for elevating intracellular polyamines during stimulation of MEPM cellular proliferation. MEPM cells treated with EGF were stimulated to proliferate and showed a dose- and time-dependent stimulation of ornithine decarboxylase (ODC) which was maximal at 4-6 hours. EGF also stimulated the initial rate of putrescine transport in a dose- and time-dependent manner. This stimulation was found to be maximal 3 hours after treatment and specific for the putrescine transport system. The kinetic parameters of putrescine transport shifted from 2.52 microM (Km) and 23.6 nmol/mg protein/15 minutes (Vmax) in nonstimulated cells to 4.48 microM (Km) and 39.8 nmol/mg protein/15 minutes (Vmax) in EGF-treated cells. This kinetic shift did not require de novo protein or RNA synthesis, as cycloheximide (10 micrograms/ml) and actinomycin D (50 micrograms/ml) had little effect on the ability of EGF to stimulate the initial rate of putrescine uptake. The rate of transport, however, was found to be inversely related to cell density. The addition of exogenous putrescine concomitantly with EGF blocked the induction of ODC, while in the presence of difluoromethylornithine (DFMO) (irreversible inhibitor of ODC) the initial rate of putrescine transport remained elevated throughout the time course studied. This stimulation of putrescine uptake caused by polyamine deprivation was reversed by exogenous putrescine and Ca++ while alpha-aminoisobutyric acid (AIB) further stimulated the rate of uptake. EGF's ability to stimulate cellular DNA synthesis was inhibited by DFMO. If DFMO-treated cells were stimulated with EGF in the presence of exogenous putrescine, this stimulatory effect was preserved. These studies indicate that the rate of polyamine transportation is highly responsive to a signal which initiates biosynthesis of polyamines. Further, this transportation system provides a compensatory mechanism allowing the cell to increase intracellular levels of polyamines when environmental conditions inhibit biosynthesis or when polyamines are abundant.     

Long-term effect of interferon on keratinocytes that maintain human papillomavirus type 31

The long-term effects of interferon treatment on cell lines that maintain human papillomavirus type 31 (HPV-31) episomes have been examined. High doses and prolonged interferon treatment resulted in growth arrest of HPV-positive cells, with a high percentage of cells undergoing apoptosis. These effects were not seen with interferon treatment of either normal human keratinocytes or cells derived from HPV-negative squamous carcinomas, which exhibited only slight decreases in their rates of growth. Within 2 weeks of the initiation of treatment, a population of HPV-31-positive cells that were resistant to interferon appeared consistently and reproducibly. The resistant cells had growth and morphological characteristics similar to those of untreated cells. Long-term interferon treatment of HPV-positive cells also resulted in a reduction in HPV episome levels but did not significantly decrease the number of integrated copies of HPV. Cells that maintained HPV genomes lacking E5 were sensitive to interferon, while cells expressing only the E6/E7 genes were resistant. In contrast, cells that expressed E2 from a tetracycline-inducible promoter were found to be significantly more sensitive to interferon treatment than parental cells. This suggests that at least a portion of the sensitivity to interferon could be mediated through the E2 protein. These studies indicate that cells maintaining HPV episomes are highly sensitive to interferon treatment but that resistant populations arise quickly.     

Epidermal Expression of Neuropilin 1 Protects Murine keratinocytes from UVB-induced apoptosis

Background

Neuropilin 1 (NRP1) is expressed on several cell types including neurons and endothelial cells, where it functions as an important regulator in development and during angiogenesis. As a cell surface receptor, NRP1 is able to bind to members of the VEGF family of growth factors and to secreted class 3 semaphorins. Neuropilin 1 is also highly expressed in keratinocytes, but the function of NRP1 in epidermal physiology and pathology is still unclear.

Methods and Results

To elucidate the role of NRP1 in skin in vivo we generated an epidermis-specific neuropilin 1 knock out mouse model by using the Cre-LoxP-System. Mice were viable and fertile and did not display any obvious skin or hair defects. After challenge with UVB irradiation, we found that deletion of epidermal NRP1 leads to increased rates of apoptosis both in vitro and in vivo. NRP1-deficient primary keratinocytes cultured in vitro showed significantly higher rates of apoptosis 24 hours after UVB. Likewise, there is a significant increase of active caspase 3 positive cells in the epidermis of Keratin 14-Cre-NRP1 (−/−) mice 24 hours after UVB irradiation. By Western Blot analysis we could show that NRP1 influences the cytosolic levels of Bcl-2, a pro-survival member of the Bcl-2 family. After UVB irradiation the amounts of Bcl-2 decrease in both protein extracts from murine epidermis and in NRP1-deficient keratinocytes in vitro, whereas wild type cells retain their Bcl-2 levels. Likewise, levels of phospho-Erk and Rac1 were lower in NRP1-knock out keratinocytes, whereas levels of pro-apoptotic p53 were higher.

Conclusion

NRP1 expression in keratinocytes is dispensable for normal skin development. Upon UVB challenge, NRP1 contributes to the prevention of keratinocyte apoptosis. This pro-survival function of NRP1 is accompanied by the maintenance of high levels of the antiapoptotic regulator Bcl-2 and by lower levels of pro-apoptotic p53.     

alpha-tocopherol increases the intracellular glutathione level in HaCaT keratinocytes

-Tocopherol is a lipophilic vitamin that exhibits an antioxidative activity. The purpose of this study was to clarify the roles of -tocopherol in the regulation of intracellular glutathione (GSH) levels in HaCaT keratinocytes. When HaCaT keratinocytes were cultivated with -tocopherol for 24 h, the intracellular GSH was increased at every concentration of -tocopherol tested. Furthermore, the HaCaT keratinocytes cultured with -tocopherol at 50 μM for 24 h exhibited resistance against H 2 O 2 . However, a short exposure of HaCaT keratinocytes to -tocopherol for 1 h did not influence either the GSH level or the resistance to H 2 O 2 . These findings suggest that GSH, which is inductively synthesized by -tocopherol, effectively reduces exogenous oxidative stress. To evaluate the effect of -tocopherol on the GSH level, BSO, which is a typical inhibitor of γ-glutamylcysteine synthetase ( γ-GCS), was used. When BSO was added to HaCaT keratinocytes, no action of -tocopherol on the GSH level was observed. On the other hand, -tocopherol resulted in the up-regulation of γ-GCS-HS (heavy subunit) mRNA. In addition, water soluble -tocopherol derivatives ( -tocopherol phosphate and trolox) caused no changes in GSH level. From these results, it was concluded that -tocopherol increases the intracellular GSH level of HaCaT keratinocytes through the up-regulation of γ-GCS-HS mRNA.     

Candida albicans actively modulates intracellular membrane trafficking in mouse macrophage phagosomes

The intracellular trafficking/survival strategies of the opportunistic human pathogen Candida albicans are poorly understood. Here we investigated the infection of RAW264.7 macrophages with a virulent wild-type (WT) filamentous C. albicans strain and a hyphal signalling-defective mutant ( efg1 Δ /cph1 Δ). A comparative analysis of the acquisition by phagosomes of actin, and of early/late endocytic organelles markers of the different fungal strains was performed and related to Candida's survival inside macrophages. Our results show that both fungal strains have evolved a similar mechanism to subvert the 'lysosomal' system, as seen by the inhibition of the phagosome fusion with compartments enriched in the lysobisphosphatidic acid and the vATPase, and thereby the acquisition of a low pH from the outset of infection. Besides, the virulent WT strain displayed additional specific survival strategies to prevent its targeting to compartmentsdisplaying late endosomal/lysosomal features, such as induction of active recycling out of phagosomes of the lysosomal membrane protein LAMP-1, the lysosomal protease cathepsin D and preinternalized colloidal gold. Finally, both virulent and efg1 Δ /cph1 Δ mutant fungal strains actively suppressed the production of macrophage nitric oxide (NO), although their cell wall extracts were potent inducers of NO.     

Epidermal growth factor receptor: Elements of intracellular communication

While EGF has an important function in cell growth regulation, the molecular mechanisms by which intracellular signal connect the EGF: receptor complex on the plasma membrane with the initiation of DNA synthesis and mitogenesis is not well understood. The discovery that rasGAP, PI-3 kinase and PLC-gamma 1 are substrates for the EGF receptor tyrosine kinase has provided a beginning in understanding the biochemistry underlying growth factor receptor transduction.     

Peptidoglycan hydrolase fusions maintain their parental specificities

The increased incidence of bacterial antibiotic resistance has led to a renewed search for novel antimicrobials. Avoiding the use of broad-range antimicrobials through the use of specific peptidoglycan hydrolases (endolysins) might reduce the incidence of antibiotic-resistant pathogens worldwide. Staphylococcus aureus and Streptococcus agalactiae are human pathogens and also cause mastitis in dairy cattle. The ultimate goal of this work is to create transgenic cattle that are resistant to mastitis through the expression of an antimicrobial protein(s) in their milk. Toward this end, two novel antimicrobials were produced. The (i) full-length and (ii) 182-amino-acid, C-terminally truncated S. agalactiae bacteriophage B30 endolysins were fused to the mature lysostaphin protein of Staphylococcus simulans. Both fusions display lytic specificity for streptococcal pathogens and S. aureus. The full lytic ability of the truncated B30 protein also suggests that the SH3b domain at the C terminus is dispensable. The fusions are active in a milk-like environment. They are also active against some lactic acid bacteria used to make cheese and yogurt, but their lytic activity is destroyed by pasteurization (63 degrees C for 30 min). Immunohistochemical studies indicated that the fusion proteins can be expressed in cultured mammalian cells with no obvious deleterious effects on the cells, making it a strong candidate for use in future transgenic mice and cattle. Since the fusion peptidoglycan hydrolase also kills multiple human pathogens, it also may prove useful as a highly selective, multipathogen-targeting antimicrobial agent that could potentially reduce the use of broad-range antibiotics in fighting clinical infections.