Preferred proline puckerings in cis and trans peptide groups: implications for collagen stability.

The interplay between side-chain and main-chain conformations is a distinctive characteristic of proline residues. Here we report the results of a statistical analysis of proline conformations using a large protein database. In particular, we found that proline residues with the preceding peptide bond in the cis state preferentially adopt a down puckering. Indeed, out of 178 cis proline residues, as many as 145 (81%) are down. By analyzing the 1-4 and 1-5 nonbonding distances between backbone atoms, we provide a structural explanation for the observed trend. The observed correlation between proline puckering and peptide bond conformation suggests a new mechanism to explain the reported shift of the cis-trans equilibrium in proline derivatives. The implications of these results for the current models of collagen stability are also discussed.

Comparison of Bcl-2 to a Bcl-2 deletion mutant for mammalian cells exposed to culture insults.

Apoptosis has been found to occur in bioreactors as a result of environmental stresses. The overexpression of bcl-2 is a widely used strategy to limit the induction of apoptosis in mammalian cell cultures. In this study, the effectiveness of wild-type Bcl-2 was compared to a Bcl-2 mutant lacking the nonstructured loop domain in two commercially prominent cell lines, Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells. The generation of a DNA "ladder" and condensation of chromatin indicated that apoptosis occurred in these cell lines following Sindbis virus infection and serum deprivation. When cells were engineered to overexpress the bcl-2 mutant, cell death due to Sindbis virus was inhibited in a concentration-dependent manner. Furthermore, the Bcl-2 mutant provided increased protection as compared to wild-type Bcl-2 following two model insults, Sindbis virus infection and serum deprivation. Total production for a heterologous protein encoded on the Sindbis virus was increased in cell lines expressing the Bcl-2 variants compared to the parental cell line. In order to understand the reasons for the improved anti-apoptosis properties of the mutant, wild-type Bcl-2 and mutant Bcl-2 were examined by Western blot following each model insult. Wild-type Bcl-2 was observed to degrade into a 23 kDa fragment following both Sindbis virus infection and serum withdrawal in both cell lines, while the mutant Bcl-2 protein was not degraded during the same period. The processing of Bcl-2 was found to correlate with reduced cell viabilities following the two external insults to suggest that Bcl-2 degradation may limit its ability to inhibit apoptosis. These studies indicate that the cells regulate anti-apoptosis protein levels and these processing events can limit the effectiveness of cell death inhibition strategies in mammalian cell culture systems.

Part I. Bcl-2 and Bcl-x(L) limit apoptosis upon infection with alphavirus vectors.

Viral expression systems offer the ability to generate high levels of a particular protein within a relatively short period of time. In particular, alphavirus constructs based on Sindbis virus (SV) and Semliki Forest virus (SFV) are promising vehicles as they are cytoplasmic vectors with the potential for high expression levels. Two such alphavirus vectors were utilized during the current study to infect two commercially relevant cell lines, baby hamster kidney (BHK) and Chinese hamster ovary (CHO); the first was a fully competent SV derivative carrying the gene for chloramphenicol acetyltransferase (dsSV-CAT), while the second was a replication deficient SFV construct containing the human interleukin-12 (IL-12) p35 and p40 genes (SFV-IL-12). Since infection with these vectors induced apoptosis in both cell lines, the present effort was dedicated to determining the ability of anti-apoptosis genes to limit the cell death associated with these virus constructs. Infection with the dsSV-CAT vector resulted in the rapid death of BHK and CHO cells within 4 days, a phenomenon which was considerably delayed by stably overexpressing bcl-2 or bcl-x(L). In fact, cellular lifespans were doubled in both BHK-bcl2 and CHO-bclx(L) cells relative to the parental cell lines. Furthermore, the presence of these gene products provided increases of up to 2-fold in recombinant CAT production. Overexpression of bcl-2 and bcl-x(L) also altered the response of these cells upon infection with SFV-IL-12. While the parental cell lines were completely nonviable within 1 week, the BHK-bcl2, BHK-bclx(L), and CHO-bclx(L) cells each recovered from the infection, resuming exponential growth and regaining viabilities of over 90% by 9 days post-infection. Total IL-12 productivities were nearly doubled by Bcl-2 and Bcl-x(L) in the CHO cells, although this effect was apparently cell-line specific, as the native BHK cells were able to secrete more IL-12 than either of its transfected derivatives. Regardless, the presence of the anti-apoptosis genes allowed the production of IL-12 to be maintained, albeit at low levels, from each of the cell lines for the duration of the culture process. Therefore, overexpression of bcl-2 family members can have a significant impact on culture viabilities and recombinant protein production during alphavirus infections of mammalian cells.

Part II. Overexpression of bcl-2 family members enhances survival of mammalian cells in response to various culture insults.

A number of bioreactor configurations have been developed for the manufacture of products from mammalian cell hosts. Even in the most efficient of these, however, problems such as nutrient exhaustion, growth factor deprivation, and toxin accumulations may arise. Consequently, the current effort focused on the feasibility of overexpressing anti-apoptosis genes in baby hamster kidney (BHK) and Chinese hamster ovary (CHO) cells as a means of limiting cell death upon exposure to three such insults. Extended periods of glucose deprivation, serum withdrawal, and treatment with ammonium chloride each caused significant damage, often apoptotic in nature, to BHK and CHO cells, typically rendering cultures completely nonviable. The overexpression of bcl-2 and bcl-x(L), however, was able to abrogate the cell death in BHK cultures, though to varying degrees. For instance, the presence of Bcl-2, which did little to suppress apoptosis upon glucose deprivation, significantly improved the viabilities of these cells during serum withdrawal. In contrast, bcl-x(L) overexpression provided BHK cells with enhanced protection in the absence of glucose, allowing cultures to remain viable throughout the entire three week study. CHO cultures, on the other hand, displayed similar trends in survival in response to both glucose and serum deprivation. During these studies, Bcl-x(L) was consistently able to afford cells the highest degree of protection, though Bcl-2 also enhanced culture viabilities and viable numbers. Death suppression following exposure to 50 mM ammonium chloride was observed to a limited extent in both BHK and CHO cells overexpressing bcl-2 and bcl-x(L). However, even during such harsh treatment, Bcl-x(L) was able to enhance the survival of both cultures, providing CHO cells with viable numbers that were nearly 20-fold that of the controls after five days of exposure. Furthermore, the extensions in cell survival provided by the anti-apoptosis gene products enabled the recovery of many of the cultures during rescue attempts in which the death-inducing stimulus was removed. Clearly, engineering cells to better withstand and recover from the insults common during the large scale cultivation of mammalian cells has a number of potential applications in the biopharmaceutical industries where cell death can limit culture productivities.

Antiapoptosis chemicals prolong productive lifetimes of mammalian cells upon Sindbis virus vector infection.

Viral expression systems allow for the rapid production of large amounts of recombinant protein in cell culture. In particular, Sindbis virus vectors now exist that make possible the expression of a variety of heterologous proteins in mammalian culture systems. Unfortunately, infection of cultured cells with Sindbis virus vectors typically results in apoptotic cell death, as demonstrated in the current study by DNA laddering and fluorescence microscopy. Fortunately, it has recently been demonstrated that apoptosis can be inhibited in vitro by certain chemical reagents that are capable of blocking specific steps during the cell death cascade. In this study, a rat prostate carcinomal cell line, AT3-neo, was infected with a Sindbis virus vector containing the gene for chloramphenicol acetyltransferase (dsSV-CAT) in the presence of several representative antiapoptotic chemicals and analyzed for cell viability as well as recombinant protein production. N-acetylcysteine (NAC), pyrrolidine dithiocarbamate (PDTC), bongkrekic acid (BA), and N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) all exhibited the capacity to limit apoptosis in the infected cells. In fact, after just 1 day, percentage viabilities of the cells exposed to chemical reagents were between 72% and 91%, compared with 44% for the untreated controls. Furthermore, cells maintained on these agents were able to survive the infection from 1 to 3 days longer than the control samples. In addition to providing gains in cell viability, chemical treatment allowed for higher levels of recombinant protein production in most cases. Maximum chloramphenicol acetyltransferase (CAT) productivities in cells maintained on BA, NAC, and Z-VAD.fmk were 1.7-, 2.2-, and 3.9-fold higher than those obtained from the untreated cultures. Consequently, the addition of chemical reagents to culture media as a means of inhibiting apoptosis may be a valuable tool in the cell culture industry, where cell death severely limits productivity levels and adds significantly to production costs.

Bcl-2 inhibits apoptosis and extends recombinant protein production in cells infected with Sindbis viral vectors.

Viruses carrying foreign genes are often used for the production of recombinant proteins in mammalian cells and other eukaryotic expression systems. Though high levels of gene expression are possible using viral vectors, the host cell generally responds to the infection by inducing apoptotic cell death within several days, abruptly ending protein production. It has recently been demonstrated, however, that apoptosis can be suppressed in virally infected cells using anti-apoptotic genes, such as bcl-2. In this study, stably transfected rat carcinomal cell lines, AT3-bcl2 and AT3-neo, were infected with a Sindbis virus carrying the gene for chloramphenicol acetyltransferase (CAT) in an effort to determine the effect of bcl-2 on cell viability and recombinant protein production. Infected AT3-bcl2 cells consistently maintained viabilities close to 100% and a growth rate equivalent to that of uninfected cells (0.040 h(-1)). In contrast, the Sindbis viral vector induced apoptosis in the AT3-neo cells, which were all dead by three days post-infection. Though infected AT3-neo cells generated higher levels of heterologous protein, over 1000 mUnits per well, CAT activity fell to zero by two days post-infection. In contrast, chloramphenicol acetyltransferase was present in AT3-bcl2 cells for almost a week, reaching a maximum level of 580 mUnits per well. In addition, recombinant protein production in AT3-bcl2 cells was extended and amplified by the regular addition of virus to the culture medium, a process which resulted in expression for the duration of the cell culture process.

Fish oil, atherogenesis, and thrombogenesis.

Marine fish consumption is known to reduce mortality from ischemic heart disease. The use of fish oil as a dietary supplement, however, is not universally recommended. In large doses, fish oil reduces plasma cholesterol and triacylglycerol but increases low density lipoprotein (LDL) levels and the potential for free radical generation and bleeding. Moderate marine fish consumption is known to reduce mortality without altering commonly measured variables, i.e., plasma cholesterol levels, in vitro platelet aggregation, and bleeding times. In swine, we observed that monocyte adhesions and platelet clumps over the lesion surface of proximal left anterior descending (LAD) coronary arteries are markedly reduced when an atherogenic diet was supplemented with cod-liver oil, even when the cholesterol levels were equalized with the untreated group. These findings suggest that fish oil is hypothrombogenic. We developed an in vitro assay to delineate the mechanism whereby fish oil reduced monocyte-endothelial cell interactions in vivo. The effects of supplementing the culture medium with different fatty acids on adhesions between lipopolysaccharide (LPS) stimulated swine aortic endothelial cells (SAEC) and the human monocyte-like cell line, U937, was investigated in a 10 minute adhesion assay at 37 degrees C. Exposure of SAEC for 6 hours to media containing 50-200 microMs eicosapentaenoic (EPA), stearic, oleic, linoleic, and arachidonic acid, respectively, revealed that only EPA reduced U937-SAEC adhesion. Exposure of U937 to EPA also reduced adhesions. EPA was not effective when added to the SAEC more than 2 hours after they were stimulated with LPS. Exposure of human umbilical vein endothelial cells (HUVEC) to EPA reduced the expression of VCAM-1, ELAM-1, and ICAM-1 after 5 hours of stimulation with LPS. These results suggest that EPA may functionally impair the induction/expression of adhesion molecules.

Observations on tumor and metastatic suppressor gene status in endometrial carcinoma with particular emphasis on p53.

BACKGROUND: Genetic changes in the development of endometrial carcinoma have not been characterized, and little is known of tumor or metastatic suppressor gene status in these malignancies. The current study on endometrioid carcinoma was undertaken to examine the status of two tumor suppressor genes that frequently have been found to be altered in human malignancies (the p53 gene and the retinoblastoma [Rb] gene) and to examine the status of the candidate metastatic suppressor gene, nm23-H1. METHODS: The status of the p53 gene was studied by immunohistochemistry of formalin-fixed paraffin-embedded biopsy samples from 72 patients with atypical endometrial hyperplasia or endometrioid carcinoma who underwent hysterectomy immediately after biopsy and from 5 patients with benign endometria. A search for loss of heterozygosity (LOH) of the nm23 gene after DNA extraction from frozen tissues and hybridization with nm23-H1 cDNA specific probe was made in 10 endometrial carcinomas. Rb gene status was evaluated by image analysis quantification of immunoreactive retinoblastoma protein in frozen sections of 10 carcinomas and 2 benign endometria. RESULTS: p53 expression was low in all benign endometria, but high expression was found in 2 (15%) of 13 atypical hyperplasias and in 23 (39%) of 59 carcinomas. High levels of p53 expression in endometrioid carcinoma correlated with the spread of disease outside of the uterus and by logistic regression, the presence of squamous differentiation, nuclear grade, and high p53 expression in the biopsy all independently correlated with spread of disease outside of the uterus. Although 7 of the 10 carcinomas studied were informative, LOH for the nm23 gene was not seen in any, including a site of metastasis. Rb protein expression in endometrial carcinoma was similar to expression in benign endometria. CONCLUSIONS: Although this study found no evidence of nm23-H1 gene alteration or alterations in Rb protein levels in endometrial carcinoma, high expression of p53 protein was sporadically identified in biopsy specimens of atypical hyperplasia and frequently found in endometrioid carcinomas. Determination of p53 expression in combination with the presence or absence of squamous differentiation and nuclear grade in biopsy material may help predict spread of endometrioid carcinoma outside the uterus and facilitate therapeutic planning before hysterectomy.