Tumor suppressor genes in the cell cycle.

In the past year, two tumor suppressor genes, retinoblastoma and p53, have been established as important players in cell-cycle control, mediated by phosphorylation and by stage-specific transcription complexes. Evidence that they also participate in other transcription complexes is accumulating and searches are underway for the downstream genes under their regulation. Genes down-regulated in tumor cells are being screened by subtractive hybridization to bridge the gap between transcription factors and their targets.

Clinical performance of scientifically designed, hot isostatic-pressed (HIP'd) zirconia cores in a bilayered all-ceramic system.

PURPOSE: The success of bilayered all-ceramic restorations is dependent upon the combination and contributions of the three principal components of these restorations: core material, core design, and core-veneer interface. The purpose of this paper is to describe the fabrication and clinical survival of optimized ceramic restorations having an explicit, scientifically designed core, machined from HIP'd isotropic zirconia and veneered using a specific protocol with thermally compatible porcelain. MATERIALS AND METHODS: Using a consistent clinical and laboratory protocol in a multicenter setting, 3,192 bilayered single and 797 bilayered splinted units were fabricated and placed on teeth and implant abutments in 1,007 patients. Approximately 61.7% (n = 2,462) were posterior restorations and 38.3% (n = 1,527) were anterior. Of the total, approximately 5.7% (n = 227) were placed on implant abutments. Survival of the restorations was determined with the Kaplan-Meier (KM) method by tooth number. RESULTS: For the 3,989 units placed, 9 failures were recorded. The KM survival of most zirconia restorations, when segregated by tooth number, was 100%. Exceptions were the 9 failed units, with a KM survival between 88% and 99% for those restorations. Six restorations failed within the first year of service, including three failed cores. Examination of those restorations revealed failure was related to initial design, quality assessment, or fabrication inconsistencies. CONCLUSION: The incorporation of a reinforcing ring beam onto an anatomically shaped core made from end-state HIP'd zirconia, in partnership with a thermally compatible veneering porcelain and a specific application protocol, resulted in extremely high survival rates for both anterior and posterior all-ceramic restorations after medium-term clinical use. These results equal or surpass the equivalent-term success rates of porcelain-fused-to-metal restorations.

Regulation of the Chlamydomonas cell cycle by light and dark.

By growing cells in alternating periods of light and darkness, we have found that the synchronization of phototrophically grown Chlamydomonas populations is regulated at two specific points in the cell cycle: the primary arrest (A) point, located in early G1, and the transition (T) point, located in mid-G1. At the A point, cell cycle progression becomes light dependent. At the T point, completion of the cycle becomes independent of light. Cells transferred from light to dark at cell cycle position between the two regulatory points enter a reversible resting state in which they remain viable and metabolically active, but do not progress through their cycles. The photosystem II inhibitor dichlorophenyldimethylurea (DCMU) mimics the A point block induced by darkness. This finding indicates that the A point block is mediated by a signal that operates through photosynthetic electron transport. Cells short of the T point will arrest in darkness although they contain considerable carbohydrate reserves. After the T point, a sharp increase occurs in starch degradation and in the endogenous respiration rate, indicating that some internal block to the availability of stored energy reserves has now been released, permitting cell cycle progression.

Adipocyte conversion of CHEF cells in serum-free medium.

When grown in the presence of serum with added insulin, Chinese hamster embryonic fibroblasts (CHEF/18) cells can be induced to become preadipocytes that are committed to the adipocyte pathway of terminal differentiation (Sager, R., and P. Kovac, 1982, Proc. Natl. Acad. Sci. USA, 79:480-484). We found that commitment to the adipocyte pathway, as well as terminal differentiation to form mature adipocytes, can occur in a defined serum-free medium containing insulin. When CHEF/18 cells are plated in serum-containing medium, only 5-10% of cells in each colony undergo terminal differentiation, whereas in serum-free medium, greater than 90% of the cells became adipocytes. These and other results show that CHEF/18 cells require no adipogenic factors in addition to insulin and the other components of the serum-free medium (transferrin, epithelial growth factor, thrombin) to form adipocytes, and furthermore, that serum inhibits the rate of terminal adipocyte differentiation of these cells. As little as 10 ng/ml insulin added to serum-containing medium can induce adipogenesis, suggesting that insulin rather than an insulinlike growth factor is the active agent. The results further demonstrate that virtually every CHEF/18 cell can be induced into the adipocyte pathway.

Specificity of gap junction communication among human mammary cells and connexin transfectants in culture.

In a previous paper (Lee et al., 1992), it was shown that normal human mammary epithelial cells (NMEC) express two connexin genes, Cx26 and Cx43, whereas neither gene is transcribed in a series of mammary tumor cell lines (TMEC). In this paper it is shown that normal human mammary fibroblasts (NMF) communicate and express Cx43 mRNA and protein. Transfection of either Cx26 or Cx43 genes into a tumor line, 21MT-2, induced the expression of the corresponding mRNAs and proteins as well as communication via gap junctions (GJs), although immunofluorescence demonstrated that the majority of Cx26 and Cx43 proteins present in transfected TMEC was largely cytoplasmic. Immunoblotting demonstrated that NMEC, NMF, and transfected TMEC each displayed a unique pattern of posttranslationally modified forms of Cx43 protein. The role of different connexins in regulating gap junction intercellular communication (GJIC) was examined using a novel two-dye method to assess homologous and heterologous communication quantitatively. The recipient cell population was prestained with a permanent non-toxic lipophilic dye that binds to membranes irreversibly (PKH26, Zynaxis); and the donor population is treated with a GJ-permeable dye Calcein, a derivative of fluorescein diacetate (Molecular Probes). After mixing the two cell populations under conditions promoting GJ formation, cells were analyzed by flow cytometry to determine the percentage of cells containing both dyes. It is shown here that Cx26 and Cx43 transfectants display strong homologous communication, as do NMEC and NMF. Furthermore, NMEC mixed with NMF communicate efficiently, Cx26 transfectants communicate with NMEC but not with NMF, and Cx43 transfectants communicate with NMF. Communication between Cx26 TMEC transfectants and NMEC was asymetrical with preferential movement of calcein from TMEC to NMEC. Despite the presence of Cx43 as well as Cx26 encoded proteins in the GJs of NMEC, few Cx43 transfectants communicated with NMEC. No heterologous GJIC was observed between Cx26- and Cx43-transfected TMEC suggesting that heterotypic GJs do not form or that Cx26/Cx43 channels do not permit dye transfer.

Transcriptional downregulation of gap-junction proteins blocks junctional communication in human mammary tumor cell lines.

Subtractive hybridization, selecting for mRNAs expressed in normal human mammary epithelial cells (NMECs) but not in mammary tumor cell lines (TMECs), led to the cloning of the human gap junction gene connexin 26 (Cx26), identified by its sequence similarity to the rat gene. Two Cx26 transcripts derived from a single gene are expressed in NMECs but neither is expressed in a series of TMECs. Northern analysis using rat Cx probes showed that Cx43 mRNA is also expressed in the normal cells, but not in the tumor lines examined. Connexin genes Cx31.1, Cx32, Cx33, Cx37, and Cx40 are not expressed in either normal cells or the tumor lines examined. In cell-cell communication studies, the normal cells transferred Lucifer yellow, while tumor cells failed to show dye transfer. Both Cx26 and Cx43 proteins were immunolocalized to membrane sites in normal cells but were not found in tumor cells. Further analysis demonstrated that Cx26 is a cell-cycle regulated gene expressed at a moderate level during G1 and S, and strongly up-regulated in late S and G2, as shown with lovastatin-synchronized NMECs. Cx43, on the contrary is constitutively expressed at a uniform low level throughout the cell cycle. Treatment of normal and tumor cells with a series of drugs: 5dB-cAMP, retinoic acid, okadaic acid, estradiol, or TGFb had no connexin-inducing effect in tumor cells. However, PMA induced re-expression of the two Cx26 transcripts but not of Cx43 in several TMECs. Thus Cx26 and Cx43 are both downregulated in tumor cells but respond differentially to some signals. Modulation of gap-junctional activity by drug therapy may have useful clinical applications in cancer.

Tumor suppressor genes: the puzzle and the promise.

Tumor suppressor genes are wild-type alleles of genes that play regulatory roles in cell proliferation, differentiation, and other cellular and systemic processes. It is their loss or inactivation that is oncogenic. The first evidence of tumor suppressor genes appeared in the early 1970s, but only within the past few years has a wealth of new information illuminated the central importance of these genes. Two or more different suppressor genes may be inactivated in the same tumors, and the same suppressors may be inactive in different tumor types (for example, lung, breast, and colon). The suppressor genes already identified are involved in cell cycle control, signal transduction, angiogenesis, and development, indicating that they contribute to a broad array of normal and tumor-related functions. It is proposed that tumor suppressor genes provide a vast untapped resource for anticancer therapy.

Measurement of fluctuating magnetic gradients originating from oceanic internal waves.

Fluctuating magnetic gradients over oceans come from electric currents flowing in seawater arising from its motions across the earth's magnetic field. Gradients of 0.3 to 0.6 picoteslas per meter for each meter of internal wave displacement have been measured at frequencies of 2 to 5 millihertz with a superconductive magnetic gradiometer supported 7 meters above the surface of water 18 meters deep about 1.5 kilometers offshore from San Diego, California.

Cytoplasmic and chloroplast ribosomes of Chlamydomonas: ultracentrifugal characterization.

Ribosomes isolated from the cytoplasmic and chloroplast fractions of Chlamydomonas were characterized in the ultracentrifuge. The cytoplasmic ribosomes belong to the 80S class of ribosomes, and, like animal ribosomes, dissociate to 60, 50, and 40S subunits. However, like the ribosomes of microorganisms, they contain smaller RNA's, 24 and 16S, and require 0.01 mole of magnesium ions per liter for stability. Chloroplast ribosomes are 70S like those of higher plants but are very unstable. A stable 50S subunit has been observed.

Maspin, a serpin with tumor-suppressing activity in human mammary epithelial cells.

A gene encoding a protein related to the serpin family of protease inhibitors was identified as a candidate tumor suppressor gene that may play a role in human breast cancer. The gene product, called maspin, is expressed in normal mammary epithelial cells but not in most mammary carcinoma cell lines. Transfection of MDA-MB-435 mammary carcinoma cells with the maspin gene did not alter the cells' growth properties in vitro, but reduced the cells' ability to induce tumors and metastasize in nude mice and to invade through a basement membrane matrix in vitro. Analysis of human breast cancer specimens revealed that loss of maspin expression occurred most frequently in advanced cancers. These results support the hypothesis that maspin functions as a tumor suppressor.

Perfusion of the human placenta with red blood cells and xanthine oxidase mimics preeclampsia in-vitro.

BACKGROUND AND PURPOSE: Preeclampsia is a major obstetric problem of unknown etiology. The fact that removal of the placenta is the only cure for preeclampsia, has led to the well-established hypothesis, that the placenta is central in the etiology. Gene profiling and proteomics studies have suggested oxidative stress caused by reperfusion and free oxygen radicals as a potential pathophysiological mechanism in preeclampsia. In this study, the dual placental perfusion model was used in order to evaluate the damaging effects of oxidative stress induced by xanthine/xanthine oxides and free hemoglobin. MATERIAL AND METHODS: The dual placenta perfusion model is a well-established in vitro model for functional placental studies. Placentas were perfused with medium containing either xanthine/xanthine oxidase or erythrocytes as a source of free hemoglobin. Concentration of free hemoglobin in the medium was measured by means of ELISA. Whole genome microarray technique and bioinformatics were used to evaluate the gene expression profile in the two groups. RESULTS: Substantial levels of free adult hemoglobin were detected in the perfusions. A total of 58 genes showed altered gene expression, the most altered were hemoglobin alpha, beta and gamma, tissue factor pathway inhibitor 2 and superoxide dismutase 2. Bioinformatics revealed that biological processes related to oxidative stress, anti-apoptosis and iron ion binding were significantly altered. CONCLUSIONS: The results suggest that perfusion with xanthine/xanthine oxidase and free hemoglobin induce changes in gene expression similar to what has been described for the preeclamptic placenta.

From the bush to the big smoke--development of a hybrid urban community based medical education program in the Northern Territory, Australia.

CONTEXT: The Northern Territory (NT) of Australia is a unique setting for training medical students. This learning environment is characterised by Aboriginal health and an emphasis on rural and remote primary care practice. For over a decade the NT Clinical School (NTCS) of Flinders University has been teaching undergraduate medical students in the NT. Community based medical education (CBME) has been demonstrated to be an effective method of learning medicine, particularly in rural settings. As a result, it is rapidly gaining popularity in Australia and other countries. The NTCS adopted this model some years ago with the implementation of its Rural Clinical School; however, urban models of CBME are much less well developed than those in rural areas. There is considerable pressure to better incorporate CBME into medical student teaching environment, particularly because of the projected massive increase in student numbers over the next few years. To date, the community setting of urban Darwin, the NT capital city, has not been well utilised for medical student training. ISSUE: In 2008, the NTCS enrolled its first cohort of students in a new hybrid CBME program based in urban Darwin. This report describes the process and challenges involved in development of the program, including justification for a hybrid model and the adaptation of a rural model to an urban setting. Relationships were established and formalised with key partners and stakeholders, including GPs and general practices, Aboriginal medical services, community based healthcare providers and other general practice and community organisations. Other significant issues included curriculum development and review, development of learning materials and the establishment of robust evaluation methods. LESSONS LEARNT: Development of the CBME model in Darwin posed a number of key challenges. Although the experience of past rural programs was useful, a number of distinct differences were evident in the urban setting. Change leadership and inter-professional collaboration were key strengths in the implementation and ongoing evaluation of the program. The program will provide important information about medical student training in urban community settings, and help inform other clinical schools considering the adoption of similar models.

Fungi and selected mycotoxins from pre- and postfermented corn silage.

AIM: To determine fungal genera, Aspergillus and Fusarium species and aflatoxin B(1) (AFB(1)), zearalenone (ZEA), deoxynivalenol (DON), fumonisin B(1) (FB(1)) contamination from pre- and postfermented corn silage produced in the most important region of Argentina where silage practice is developed. METHODS AND RESULTS: Sampling of corn silos was performed manually through silos in transects at three levels: upper, middle and low sections. AFB(1) and FB(1) were quantified by high-performance liquid chromatography, zearalenone by enzyme-linked immunosorbent assay and DON by gas chromatography. Over 90% of the samples showed counts higher than 1 x 10(4) CFU g(-1). Aspergillus flavus and Fusarium verticillioides were the prevalent species. Some tested samples were contaminated with AFB(1), ZEA, DON and FB(1). CONCLUSIONS: This study demonstrates the presence of fungi and AFB(1), ZEA, DON and FB(1) contamination in corn silage in Argentina. SIGNIFICANCE AND IMPACT OF THE STUDY: This manuscript makes a contribution to the knowledge of mycotoxins in Argentinean silage in particular because the environmental conditions in this country differ from those of most reports. The comparison of pre- and postfermentation silage is also outstanding. Therefore, information on fungi and mycotoxins present in silage--an increasingly popular commodity--is useful to estimate potential risk for animal and human health.

Dual in vitro perfusion of an isolated cotyledon as a model to study the implication of changes in the third trimester placenta on preeclampsia.

In the current study perfusions of an isolated cotyledon of term placenta using standard medium were compared to medium containing xanthine plus xanthine oxidase (X+XO), which generates reactive oxygen species (ROS). A time-dependant increase in the levels of different cytokines (TNF-alpha, IL-1ss, IL-6, IL-8 and IL-10) was observed between 1 and 7h with more than 90% of the total recovered from the maternal compartment with no significant difference between the 2 groups. For 8-iso-PGF2alpha 90% of the total was found in the fetal compartment and a significantly higher total release was seen in the X+XO group. Microparticles (MPs) isolated from the maternal circuit were identified by flow cytometry as trophoblastic sheddings, whereas MPs from the fetal circuit were predominantly derived from endothelial cells. More than 90% of the total of MPs was found in the maternal circuit. The absolute amount of the total as well as the maternal fraction were significantly higher in the X+XO group. Immunohistochemistry (IHC) of the perfused tissue revealed staining for IL-1beta of villous stroma cells, which became clearly more pronounced in experiments with X+XO. Western blot of tissue homogenate revealed 2 isoforms of IL-1beta at 17 and 31kD. In X+XO experiments there was a tendency for increased expression of antioxidant enzymes in the tissue. Western blot of MPs from the maternal circuit showed increased expression of antioxidant enzymes in the X+XO group and for IL-1beta only the 17kD band was detected. In vitro reperfusion of human placental tissue results in mild tissue injury suggestive of oxidative stress. In view of the increased generation of ROS in perfused tissue with further increase under the influence of X+XO, the overall manifestation of oxidative stress remained rather mild. Preservation of antioxidant capacity of human placental tissue could be a sign of integrity of structure and function being maintained in vitro by dual perfusion of an isolated cotyledon. The observed changes resemble findings seen in placentae from preeclampsia.

Loss of chloroplast DNA methylation during dedifferentiation of Chlamydomonas reinhardi gametes.

In Chlamydomonas reinhardi the chloroplast DNA (ch;DNA) of mating type plus cells undergoes cyclical methylation and demethylation during the life cycle. Methylation occurs during gametogenesis, and fully differentiated gametes can be dedifferentiated back to vegetative cells which contain nonmethylated chlDNA by the addition of a nitrogen source for growth. We examined the dedifferentiation process and found that the mating ability of gametes was lost rapidly after the start of dedifferentiation at a time when the chlDNA was still methylated. The enzymatic activity of the 200-kilodalton DNA methyltransferase was lost at a rate consistent with the rate of dilution during cell division. Methylation of chlDNA decreased at a slower rate than was expected from cell division alone but was consistent with the continuing activity of the preexisting methyltransferase so long as it was present. These results support the hypothesis that demethylation of chlDNA occurs by dilution out of enzymatic methylating activity rather than by enzymatic demethylation.

C-reactive protein production in term human placental tissue.

OBJECTIVE: C-reactive protein (CRP) is a marker of systemic inflammation. Recently, it has been shown that CRP is present in amniotic fluid and fetal urine, and that elevated levels are associated with adverse pregnancy outcome. However, the precise source of amniotic fluid CRP, its regulation, and function during pregnancy is still a matter of debate. The present in vivo and in vitro studies were designed to investigate the production of CRP in human placental tissues. MATERIAL AND METHODS: Ten paired blood samples from peripheral maternal vein (MV), umbilical cord artery (UA) and umbilical vein (UV) were collected from women with elective caesarean sections at term. The placental protein accumulation capacity of hCG, hPL, leptin and CRP was compared with the dual in vitro perfusion method of an isolated cotyledon of human term placentae and quantified by ELISA. Values for accumulation (release) were calculated as total accumulation of maternal and fetal circuits normalized for tissue weight and duration of perfusion. For gene expression, RNA was extracted from placental tissue and reverse transcribed. RT-PCR and real-time PCR were performed using specific primers. RESULTS: The median (range) CRP level was significantly different between UA and UV [50.1 ng/ml (12.1-684.6) vs. 61 ng/ml (16.9-708.1)]. The median (range) difference between UV and UA was 9.3 ng/ml (2.2-31.6). A significant correlation was found between MV CRP and both UA and UV CRP levels. Median (range) MV CRP levels [2649 ng/ml (260.1-8299)] were 61.2 (6.5-96.8) fold higher than in the fetus. In vitro, the total accumulation rates (mean+/-SD) were 31+/-13 (mU/g/min, hCG), 1.16+/-0.19 (microg/g/min, hPL), 4.71+/-1.91 (ng/g/min, CRP), and 259+/-118 (pg/g/min, leptin). mRNA for hCG, hPL and leptin was detectable using conventional RT-PCR, while CRP mRNA could only be demonstrated by applying real-time RT-PCR. In the perfused tissue the transcript levels for the four proteins were comparable to those detected in the native control tissue. CONCLUSIONS: Our results demonstrate that the human placenta produces and releases CRP mainly into the maternal circulation similarly to other analyzed placental proteins under in vitro conditions. Further studies are needed to explore the exact role of placental CRP during pregnancy.

Multistep origin of tumor-forming ability in Chinese hamster embryo fibroblast cells.

Twenty-one anchorage-independent subclones and ten subclones with reduced serum requirements were isolated as single-step mutants spontaneously or after ethylmethanesulfonate or N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis of CHEF/18 diploid Chinese hamster embryo fibroblasts. Anchorage-independent mutants retain the high serum requirement and nontransformed morphology typical of CHEF/18. Only four of 21 anchorage mutants have spontaneously produced tumors when injected at 10(7)/site in nude mice, and these were only at a fraction of sites. Low-serum (LS) mutants acquire transformed morphology and increased anchorage-independent growth simultaneously with the loss of high-serum requirement. Only two of ten LS mutants have spontaneously produced tumors. However, when some anchorage and LS mutants were remutagenized and when mutagenized populations were injected into nude mice, tumors appeared at many of the injected sites. In contrast, untreated CHEF/18 cells have never given tumors(0 of 34 sites), and mutagenized DHEF/18 cells have given tumors at only three of 29 sites. These results demonstrate that malignant transformation is a multistep process in the Chinese hamster embryo fibroblast system. Most one-step mutants selected for anchorage independence or reduced serum requirements do not have tumor-forming potentials higher than that of the parent CHEF/18. Thus anchorage-independent and LS phenotypes per se do not account for the increase in tumor-forming potential. It is proposed that the genomic rearrangement process as well as specific mutations may contribute to tumorigenicity.

Estrogen inhibits the growth of estrogen receptor-negative, but not estrogen receptor-positive, human mammary epithelial cells expressing a recombinant estrogen receptor.

Estrogen is essential for the growth of the normal mammary gland and most estrogen receptor (ER)-positive mammary carcinomas. To better understand the differences between the estrogen response pathways in normal and tumor cells, we have stably transfected ER-negative immortal, nontumorigenic human mammary epithelial cells and ER-negative breast cancer cells with an ER-encoding expression vector. Unexpectedly, estrogen treatment (1.0 nM) inhibited the proliferation of ER-transfected nontumorigenic and tumor-derived cells. The control transfectants and parental cells exhibited no response to estrogen concentrations as high as 1.0 microM. This inhibitory effect was attributed to a decreased growth rate and a perturbation of the cell cycle distribution by estrogen treatment of the ER transfectants. The inhibitory response was blocked by cotreatment with the antiestrogen ICI 164,384 as predicted for a pure antagonist of estrogen action. However, treatment with the antiestrogen hydroxytamoxifen caused growth inhibition, implying that hydroxytamoxifen acts as an agonist of estrogen action in ER-transfected cells. Since estrogen is a mitogenic and not a growth-inhibitory stimulus for ER-positive breast cancers and cell lines, we tested the effect of constitutive, high level expression of the ER in ER-positive tumor cells. Stable transfection of ER-positive MCF-7 and T47D cells with the ER expression vector yielded cells with varying amounts of ER. At ER levels comparable to those found in the ER-negative transfected cells, the MCF-7 and T47D ER transfectants were not inhibited by estrogen. These data suggest that ER-positive breast cancer cells can tolerate higher constitutive levels of ER expression than ER-negative cells. The mechanism by which this is accomplished may be an essential step in the process which yields ER-positive tumors.