Higher Saturation Density Research Articles

The Link between Autosomal Dominant Polycystic Kidney Disease and Chromosomal Instability: Exploring the Relationship.

In autosomal dominant polycystic kidney disease (ADPKD) with germline mutations in a PKD1 or PKD2 gene, innumerable cysts develop from tubules, and renal function deteriorates. Second-hit somatic mutations and renal tubular epithelial (RTE) cell death are crucial features of cyst initiation and disease progression. Here, we use established RTE lines and primary ADPKD cells with disease-associated PKD1 mutations to investigate genomic instability and DNA damage responses. We found that ADPKD cells suffer severe chromosome breakage, aneuploidy, heightened susceptibility to DNA damage, and delayed checkpoint activation. Immunohistochemical analyses of human kidneys corroborated observations in cultured cells. DNA damage sensors (ATM/ATR) were activated but did not localize at nuclear sites of damaged DNA and did not properly activate downstream transducers (CHK1/CHK2). ADPKD cells also had the ability to transform, as they achieved high saturation density and formed colonies in soft agar. Our studies indicate that defective DNA damage repair pathways and the somatic mutagenesis they cause contribute fundamentally to the pathogenesis of ADPKD. Acquired mutations may alternatively confer proliferative advantages to the clonally expanded cell populations or lead to apoptosis. Further understanding of the molecular details of aberrant DNA damage responses in ADPKD is ongoing and holds promise for targeted therapies.

Fluctuation of Satellite Charging in the Low Earth Orbit Regime due to Interaction with Ionospheric Plasma

Low Earth-orbiting satellites will always interact with the ionospheric plasma, leading to disruption of satellite operations, depending on the impact of the interaction. Under normal circumstances (without interference), the magnitude of the fluctuation in satellite charging is less than 5V (negative). Any disturbance from charged particles will cause the satellite to experience a more drastic voltage drop of the order of a few hundred volts (negative). The identification of fluctuations in satellite charging is carried out by reviewing the space conditions around the satellite, characterized by several parameters such as density, energy, and plasma temperature, as well as the saturation density of the satellite. To strengthen the analysis, this study deepens the flux changes with an energy of the order of MeV along the satellite orbit. The results of the study show that several low-earth orbit satellites experience a fairly high saturation density, which is correlated with fluctuations in electron flux in some regions traversed by the satellite, such as the polar and the SAA regions. The results confirm that the two regions could be a danger zone for both polar and equatorial satellites.

Elucidation of the mechanism underlying CD44v6-induced transformation of IEC-6 normal intestinal epithelial cells.

The transformation abilities of CD44s and CD44v6 in normal intestinal epithelial cells have not yet been reported. Herein, we established both CD44sand CD44v6overexpressing stable clones from rat IEC-6 cells and demonstrated that the CD44v6 clones had higher saturation density and anchorage independence. Additionally, CD44v6 clones were more resistant to oxaliplatin and irinotecan which might be attributed to a significantly increased B-cell lymphoma 2 level and a reduced DNA damage response in these cells. Moreover, c-Met and vascular endothelial growth factor receptor 2 signalings were involved in modulating the saturation density in CD44v6 clones. Interestingly, higher activation of both AKT and extracellular-signal-regulated kinase (ERK) were detected in CD44v6 clones which might account in part for the cell density-independent nuclear localization of Yes-associated protein (YAP). To no surprise, increases of both saturation density and anchorage independence in CD44v6 clones were markedly diminished by PI3K, AKT, MEK, and ERK inhibitors as well as YAP knockdown. By contrast, overexpression of a constitutively active YAP robustly increased the aforementioned phenotypes in IEC-6 cells. Collectively, our results suggest that upregulation of CD44v6, but not CD44s, induces the transformation of normal intestinal epithelial cells possibly via activating the c-Met/AKT/YAP pathway which might also explain the important role of CD44v6 in the initiation of various carcinomas.

Development of soft magnetic amorphous alloys with distinctly high Fe content

This paper reports on the preparation of Fe82.7-85.7Si2-4.9B9.2-11.2P1.5-2.7C0.8 soft magnetic amorphous alloys with a distinctly high Fe content of 93.5-95.5 wt.% by component design and composition adjustment. All alloys can be readily fabricated into completely amorphous ribbon samples with good surface quality by the single copper roller melt-spinning method. These alloys show good bending ductility and excellent magnetic properties after annealing, i.e., low coercivity ( H c) of 3.3-5.9 A/m, high permeability ( μ e) of 5000-10000 and high flux saturation density ( B s) of 1.63-1.66 T. The mechanism of the good glass forming ability (GFA) and soft-magnetic properties are explored. The amorphous alloys with the high Fe content comparable to that of the desired high Si alloy can be promising candidates for the potential application in electric devices.

Use of Antibiotics for Maintenance of Axenic Cultures of Amphidinium carterae for the Analysis of Translation.

Most dinoflagellates in culture are bacterized, complicating the quantification of protein synthesis, as well as the analysis of its regulation. In bacterized cultures of Amphidinium carterae Hulbert, up to 80% of protein synthetic activity appears to be predominantly bacterial based on responses to inhibitors of protein synthesis. To circumvent this, axenic cultures of A. carterae were obtained and shown to respond to inhibitors of protein synthesis in a manner characteristic of eukaryotes. However, these responses changed with time in culture correlating with the reappearance of bacteria. Here we show that culture with kanamycin (50 μg/mL), carbenicillin (100 μg/mL), and streptomycin sulfate (50 μg/mL) (KCS), but not 100 units/mL of penicillin and streptomycin (PS), prevents the reappearance of bacteria and allows A. carterae protein synthesis to be quantified without the contribution of an associated bacterial community. We demonstrate that A. carterae can grow in the absence of a bacterial community. Furthermore, maintenance in KCS does not inhibit the growth of A. carterae cultures but slightly extends the growth phase and allows accumulation to somewhat higher saturation densities. We also show that cultures of A. carterae maintained in KCS respond to the eukaryotic protein synthesis inhibitors cycloheximide, emetine, and harringtonine. Establishment of these culture conditions will facilitate our ability to use polysome fractionation and ribosome profiling to study mRNA recruitment. Furthermore, this study shows that a simple and fast appraisal of the presence of a bacterial community in A. carterae cultures can be made by comparing responses to cycloheximide and chloramphenicol rather than depending on lengthier culture-based assessments.

Pigmented Ink Technology to Enable HP PageWide XL Printer Capability

The recently announced HP Pagewide XL printer delivers high quality output at very high printing speeds. The breakthrough speed of Pagewide XL is delivered via an array of nozzles covering the entire paper width (an order of magnitude more nozzles than traditional scanning systems). All the drops are fired in a single pass, which leads to a heightened sensitivity to nozzle errors for output quality, and to servicing drops for print production costs. Robust and reliable operation, along with economical print production are enabled by an inkset developed for this application. First, the Pagewide XL inkset uses pigment colorants that are quickly immobilized when contacting paper surface, leading to high color saturation and high black optical density at lower levels of ink coverage. The pigmented colorants, along with HP's polymeric binders also deliver better resistance to water, highlighters, and light fade. Second, the inkset has an additive that migrates to the ink/air interface in the nozzle. This film-forming additive produces a vapor barrier that slows water loss and enables extended uncapped periods while maintaining good drop ejection. Finally, the ink components work together to minimize the need for servicing spits to keep nozzles healthy, which improves uptime, productivity, and reduces print production costs.

The paradox of the contrasting roles of chronic magnesium deficiency in metabolic disorders and field cancerization.

Magnesium (Mg(2+)) deficiency is common in metabolic disorders such as obesity, type 2 diabetes, and insulin resistance. These disorders are also associated with a high incidence of cancer. Mg(2+) is the regulator par excellence of metabolism, largely through its role as a cofactor for all phosphoryl transfers in the cell. Because Mg(2+) deficiency inhibits energy production it might be expected to inhibit tumor production. However, the high incidence of cancer in metabolic disorders makes that seem unlikely. In order to understand this seeming paradox, it is important to understand the regulatory role of Mg(2+) in normal and neoplastic cells. Free Mg(2+) is the primary regulator of glycolysis and the Krebs cycle. It also acts as a second messenger for growth factors in regulating protein synthesis. Varying Mg(2+) concentrations result in the same set of coordinated responses as varying serum concentrations. Selection by serial rounds of high cell density or reduced serum concentration at low cell density results in progressive stages of field cancerization. Highly transformed cells proliferate in much lower concentrations of Mg(2+) and grow to much higher saturation densities than normal cells. It remains to be seen whether reduction in Mg(2+) in sparse, exponentially proliferating cultures selects for increases in saturation density and transformed foci.

P21 Waf1 is required for complete oncogenic transformation of mouse embryo fibroblasts by E1Aad5 and c-Ha-ras oncogenes

Cyclin-dependent kinase inhibitor p21 Waf1 is known to have alternative functions associated with positive regulation of proliferation, actin cytoskeleton remodeling and suppression of apoptosis. The goal of the present study was to assess the role of p21 Waf1 in the establishment of the transformed phenotype of mouse embryo fibroblasts with stable expression of E1Aad5 and c-Ha-ras complementary oncogenes. Herein, we demonstrate that E1A/c-Ha-Ras-transformed p21 Waf1-null fibroblasts possess some characteristic features of transformed cells, such as loss of contact inhibition, high saturation density, shortened cell cycle, inability to undergo cell-cycle arrest after DNA damage and serum deprivation, but, at the same time, they are not completely transformed in that they are unable to proliferate at clonal density, are anchorage-dependent, retain a fibroblast-like morphology with pronounced actin cytoskeleton and show reduced migration and invasion. Our data support the concept of p21 Waf1 “tumor suppressor” having alternative oncogenic functions in the cytoplasm and for the first time indicate that p21 Waf1 can be indispensable for complete oncogenic transformation.

Minimizing oxygen inclusion when electroplating high saturation density CoFe for microelectromechanical system

Co–Fe alloys with a high saturation flux density Bs are used as soft magnetic materials in various microelectromechanical system applications. The electroplating process to deposit this material was widely researched. This paper describes experimental investigations to optimize the Co–Fe electroplating process. Besides the high saturation flux density Bs and other magnetic properties, mechanical and chemical properties like low film stress and good corrosion resistance also have to be considered. In these experiments, test electrolytes with different additives were used. The main purpose of those additives is to avoid the oxidation of Fe2+ to Fe3+ in the electrolyte to minimize the formation of Fe(OH)3 and its integration in the deposited film. Vibrating sample magnetometer measurements were applied to characterize the magnetic properties. The composition of Fe and Co in the deposited film was determined by energy dispersive x-ray spectroscopy. An electron probe microanalyzer was used to determine the impurities (O, S, and B) in the deposits. The results show different efficiencies of the applied additives improving the plating process.

Abstract A34: Curcumin downregulates IL-8 in ER negative DCIS breast cancer model: Correlation between HER-2-induced inflammation and tumorigenecity

Abstract A34 Introduction Overexpression of HER-2 oncogene is reported in more than 60% of ductal carcinoma in situ (DCIS) breast cancer, including 30% estrogen receptor negative chemotherapy resistant high-grade tumors with metastatic phenotype. Recent studies show high correlation between MRI characteristics and lower ER score, higher grade, much higher CD68 count (inflammatory marker) and extensive comedo necrosis of DCIS. The inflammatory cytokine IL-8 is found to be overexpressed in most ER- breast cancers and is recently suggested as a marker of tumor invasion and metastasis. Curcumin (CUR) the active component of the spice turmeric, known for its immunomodulatory, antimutagenic and antitumorigenic properties and was found to inhibit cell proliferation and mitogenic signal transduction pathways in ER negative, HER-2 positive DCIS breast cancer model. Material and Methods Normal 184-B5 breast epithelial cells and transformed 184-B5/HER cells in log phase were exposed to 0, 5, 10 and 20 μM CUR for 24 and 48 hours and analyzed for dose response, cell viability, cell cycle distribution, gene expression and HER-2 signal transduction protein markers. Results 184-B5/HER cells exhibit hyper-proliferation, shorter population doubling time, higher saturation density, reduced rate of apoptosis, higher anchorage independent growth and tumorigenecity compared to the non-tumorigenic ER-, PR-, HER2-, EGFR+ and p53+ parental immortalized 184-B5 cells. Cell cycle analysis of 184-B5/HER cells exposed to 20μM CUR showed cytostatic arrest of growth, 85% inhibition of colony formation, decreased G0/G1: S+G2/M ratio and increased apoptosis. Microarray data showed 557 probes to be modulated in 20μM CUR (24 hrs) treated 185-B5/HER cells (117 upregulated, 380 downregulated). These include reduced expression of cell cycle regulatory genes such as cyclin A (-2.79), cyclin B2 (-3.89), cyclin D1 (-1.50), cdk2 (-1.69) and Cdc25c (-3.80). Decreased levels of Cyclin B2, cdk2 and Cdc25c cell cycle proteins in 184-B5/HER cells, reflect CUR induced cell cycle arrest in S and G2/M phases. Treatment with CUR decreased NFkB (-1.86) in 184-B5/HER cells suggesting modulation of downstream signaling pathways including the inflammatory cascade. We observed a 10 fold higher levels of interleukin-8 (IL-8) gene expression in 184-B5/HER cells compared to 184-B5 cells, while treatment with CUR dramatically reduced the IL-8 gene expression as revealed by both microarray and real-time PCR. Conclusion Treatment of breast epithelial 184-B5/HER cells with CUR show preventive efficacy in this ER negative DCIS model via cytostatic arrest in S and G2/M phases of the cell cycle, by downregulation of ERK pathway and induction of apoptosis, verified at both the gene and protein levels. Moreover, CUR down-regulates key regulatory proteins such as NFkB and IL-8 expression thus preventing the inflammatory cascade and possible progression of DCIS into invasive ductal carcinoma. Reduced expression of metastatic molecular marker IL-8 by CUR in 184-B5/HER cells suggests its preventive/therapeutic potential [Support: NIH-NCI CA122394]. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A34.

Malignant Transformation of Normal Enterocytes following Downregulation of Bak Expression

Bak is a pro-apoptotic gene, which plays an important role in the multi-step process of gastrointestinal tumorigenesis. We hypothesized that downregulation of Bakexpression in normal enterocytes will result in a transformed phenotype. The nontumorigenic intestinal epithelial cell line (IEC18) was transfected with the vector pMV12-AS-bak (encoding anti-sense bak). Three clones, with Bakprotein levels similar to those seen in colon cancer cell lines and significantly lower than those found in the parental cells, were further evaluated. The three clones proliferated faster, demonstrated anchorage-independent growth in soft agar and a higher saturation density and plating efficiency. Furthermore, when injected into nude mice, these cells generated tumors after approximately 2–3 weeks. The cells were more resistant to the induction of apoptosis by sulindac sulfide and sulindac sulfone but more sensitive to COX 2 inhibitors (celecoxib and nimesulide). The levels of p16, cyclin D1 and COX 2 were higher in the three transformed clones. In summary,downregulation of Bak expression in normal enterocytes contributes to abnormal growth and tumorigenesis. COX 2 inhibitors may serve as important agents in the prevention and treatment of CRC as they only inhibit the growth of malignant cells.

Donor binding energy and thermally activated persistent photoconductivity in high mobility (001) AlAs quantum wells

A doping series of AlAs (001) quantum wells with Si δ-modulation doping on both sides reveals different dark and postillumination saturation densities, as well as temperature dependent photoconductivity. The lower dark two-dimensional electron density saturation is explained assuming deep binding energy of ΔDK=65.2meV for Si donors in the dark. Persistent photoconductivity (PPC) is observed upon illumination, with higher saturation density indicating shallow postillumination donor binding energy. The photoconductivity is thermally activated, with 4K illumination requiring postillumination annealing to T=30K to saturate the PPC. Dark and postillumination doping efficiencies are reported.

Reduced Levels of ATF-2 Predispose Mice to Mammary Tumors

Transcription factor ATF-2 is a nuclear target of stress-activated protein kinases, such as p38, which are activated by various extracellular stresses, including UV light. Here, we show that ATF-2 plays a critical role in hypoxia- and high-cell-density-induced apoptosis and the development of mammary tumors. Compared to wild-type cells, Atf-2(-/-) mouse embryonic fibroblasts (MEFs) were more resistant to hypoxia- and anisomycin-induced apoptosis but remained equally susceptible to other stresses, including UV. Atf-2(-/-) and Atf-2(+/-) MEFs could not express a group of genes, such as Gadd45alpha, whose overexpression can induce apoptosis, in response to hypoxia. Atf-2(-/-) MEFs also had a higher saturation density than wild-type cells and expressed lower levels of Maspin, the breast cancer tumor suppressor, which is also known to enhance cellular sensitivity to apoptotic stimuli. Atf-2(-/-) MEFs underwent a lower degree of apoptosis at high cell density than wild-type cells. Atf-2(+/-) mice were highly prone to mammary tumors that expressed reduced levels of Gadd45alpha and Maspin. The ATF-2 mRNA levels in human breast cancers were lower than those in normal breast tissue. Thus, ATF-2 acts as a tumor susceptibility gene of mammary tumors, at least partly, by activating a group of target genes, including Maspin and Gadd45alpha.

Tensin2 variant 3 is associated with aggressive tumor behavior in human hepatocellular carcinoma†

Tensins are a new family of proteins that act as an important link among extracellular matrix, actin cytoskeleton, and signal transduction and have been implicated in human cancers. Tensin2 was initially identified in a search for new tensin family members that share extensive sequence homology with tensin1. Tensin2 was highly expressed in liver tissues. A recent study reported that one of the splicing variants of tensin2, variant 3, promotes cell migration. In the present study, we aimed to elucidate the role of variant 3 in hepatocarcinogenesis by assessing the expression of variant 3 mRNA in hepatocellular carcinoma (HCC) tissue and ectopically expressing variant 3 in HCC cell lines. Analysis of variant 3 expression in human HCC tissue revealed it was overexpressed in 46% (23/50) of tumor tissues as compared with the corresponding nontumorous livers. High expression of variant 3 was significantly associated with venous invasion (P = .037), tumor microsatellite formation (P = .022), and tumor nonencapsulation (P = .049). Our ectopic expression study showed that variant 3 significantly promoted the cell growth and motility of HCC cells. The clonal transfectants of variant 3 were more closely packed and resulted in a higher saturation density than in the control vector transfectants. Variant 3 expression also enhanced the proliferation rate in culture and in vivo tumorigenicity in nude mice. In conclusion, we reveal a novel role for variant 3 in the progression of HCC and suggest the feasibility of elevated variant 3 expression as a tumor progression marker for HCC.

Epstein-Barr virus (EBV)-encoded RNA promotes growth of EBV-infected T cells through interleukin-9 induction.

EBV associates with various T-cell-proliferating diseases such as chronic active EBV infection and nasal lymphoma. In contrast to B cells, which are highly susceptible to EBV infection in vitro, T cells are refractory to EBV infection in vitro, and it has been difficult to examine the effects of EBV infection on T cells. We recently generated EBV recombinants with a selectable marker, which made it possible to select EBV-infected cells even when the efficiency of infection was low. Using the recombinant virus, we found that a human T-cell line, MT-2, was susceptible to EBV infection, and we succeeded in isolating EBV-infected cell clones with type II EBV latency, which was identical with those seen in EBV-infected T cells in vivo. EBV-infected MT-2 cell clones had shorter cell doubling times and higher saturation density than non-EBV-infected counterparts. We found that EBV-positive MT-2 cells expressed higher levels of interleukin (IL)-9 than EBV-negative MT-2 cells at the transcriptional level. It was also demonstrated that EBV-encoded small RNA was responsible for IL-9 expression. Addition of recombinant IL-9 accelerated the growth of MT-2 cells, whereas growth of the EBV-converted MT-2 cells was blocked by treatment with an anti-IL-9 antibody. These results suggest that IL-9 induced by EBV-encoded small RNA acts as an autocrine growth factor for EBV-infected T cells. Analysis of nasal lymphoma biopsies indicated that three of four specimens expressed IL-9. The present findings suggest that EBV directly affects the pathogenesis of EBV-associated T-cell diseases.

Bovine Leukemia Virus Alters Growth Properties and Casein Synthesis in Mammary Epithelial Cells

Bovine leukemia virus (BLV) is widespread in US dairy herds, yet only about 1% of infected cattle develop bovine leukosis and are culled from the herd. A major concern is whether BLV infection of dairy cows alters milk yield. Although several studies have examined the effect of BLV on milk production in vivo, the results were inconclusive. No in vitro studies have been done. The discovery of BLV in mammary epithelial cells (MEC) of infected cows raises the possibility that the virus could affect these cells directly. The purpose of this study was to use an in vitro system to determine if BLV could alter milk yield by altering cell number and/or milk production per cell. A short-term cell line established from the MEC of a BLV-negative cow, and a proven casein-producer mouse cell line, Comma D, were stably transfected with a plasmid containing the entire BLV genome. Untransfected parental lines served as negative controls. The BLV-containing bovine MEC line has a reduced population-doubling time, higher saturation density, and increased longevity. The Comma D line is an already-transformed cell line, and growth properties did not change after transfection with BLV. Under appropriate differentiation conditions, both the bovine and mouse MEC transfected with BLV displayed decreased casein production and mRNA synthesis compared with control cell lines without BLV. Our results suggest that effects of BLV infection on milk production may not be related solely to overall animal health but may also be mediated directly at a cellular level.

Overexpression of the 18 kDa and 22/24 kDa FGF-2 isoforms results in differential drug resistance and amplification potential.

We investigated the role of low molecular weight (LMW) and high molecular weight (HMW) isoforms of basic fibroblast growth factor 2 (FGF-2) in the expression of transformation-related phenotypic alterations, drug sensitivity modulation, and gene amplification potential. For this purpose, we used NIH 3T3 and A31 cells transfected with different cDNA FGF-2 constructs allowing expression of the different proteins. Both cell lines showed marked phenotypic alterations when expressing the LMW FGF-2 or the four HMW FGF-2 isoforms: they acquired a transformed morphology, grew at higher saturation densities in 10% serum, and exhibited anchorage-independent growth and increased invasive potential. However, HMW FGF-2-expressing cells also grew in 1% serum and their invasive potential was lower than in cells expressing all FGF-2 forms or LMW FGF-2 alone. We have grown the different cell lines under a selective pressure of N-(phosphonacetyl)-l-aspartate (PALA), a drug which specifically inhibits the aspartate transcarbamylase activity of the multifunctional carbamyl-P-synthetase/aspartate transcarbamylase/dihydro-orotase genes (CAD) enzyme (and thus inhibits de novo pyrimidine biosynthesis) and selects for cells with amplified copies of the CAD gene. Our results demonstrate that aberrant expression of the LMW FGF-2 and/or HMW FGF-2 isoforms differently modulates drug resistance and gene amplification properties in the NIH 3T3 and A31 cell lines by differential amplification of the CAD gene. Coexpression of all isoforms appears to be necessary to obtain cumulative effects and nuclear-targeted HMW FGF-2 has a pivotal role in such a cooperation.

Ras-inducible immortalized fibroblasts: focus formation without cell cycle deregulation.

The Ras oncogene transforms cultured murine fibroblasts into malignant, focus-forming cells, whose lack of contact inhibition is evidenced by high saturation densities. In order to investigate the reversibility of Ras transformation, as well as the kinetics of Ras-induced changes, cell lines that conditionally express oncogenic Ras were constructed. Both focus formation and increased saturation density were inducible and fully reversible. In exponentially growing cells, oncogenic Ras-expression had no effect on proliferation rates, Erk phosphorylation, or the level of cyclin D1, and Ras-induction did not confer serum-independent growth. As expected, growth to high density in uninduced cells led to quiescence with a low level of cyclin D1 and no active Erk; in this setting, Ras induction prevented full downregulation of cyclin D1 and inactivation of Erk. Our results show that Ras expression to a level sufficient for transformation leads to relatively subtle effects on known downstream targets, and that the focus formation and increased saturation density growth induced by Ras is not a result of growth factor independence.

The Brn-3b POU family transcription factor regulates the cellular growth, proliferation, and anchorage dependence of MCF7 human breast cancer cells.

The Brn-3b POU domain containing transcription factor is expressed in the developing sensory nervous system as well as in epithelial cells of the breast, cervix, and testes. Brn-3b functionally interacts with the estrogen receptor (ER) and in association with the ER, regulates transcription from estrogen responsive genes. In addition, Brn-3b expression is elevated in breast tumours compared to levels in normal mammary cells. To explore the role of Brn-3b in breast cancer, we established stable cell lines derived from the MCF7 human breast cancer cell line which had been transfected with Brn-3b sense or anti-sense constructs. The Brn-3b over-expressing cell lines exhibited increased growth rate, reached confluence at a higher saturation density, had higher proliferative activity, and an enhanced ability to form colonies in soft agar when compared to the control empty vector transfected cells. Likewise, the Brn-3b anti-sense cell lines showed reduced cellular growth and proliferation, reached confluence at a lower density, and exhibited a decreased ability to form colonies in soft agar when compared to the vector controls. Five to ten per cent of the Brn-3b over-expressing cells exhibited a severely altered morphology characterized by reduced adherence to tissue culture plastic, increased cell size, and a vacuolar cell shape. These results thus further indicate a role for the Brn-3b transcription factor in regulating mammary cell growth and suggest that its elevation in breast cancer is of functional significance.

Activation of the Rap1 Guanine Nucleotide Exchange Gene,CalDAG-GEF I, in BXH-2 Murine Myeloid Leukemia

Here we report the recurrent proviral activation of the Rap1-specific guanine nucleotide exchange factor CalDAG-GEF I (Kawasaki, H., Springett, G. M., Toki, S., Canales, J. J., Harlan, P., Blumenstiel, J. P., Chen, E. J., Bany, I. A., Mochizuki, N., Ashbacher, A., Matsuda, M., Housman, D. E., and Graybiel, A. M. (1998) Proc. Natl. Acad. Sci. U. S. A. 95, 13278-13283; Correction (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 318) gene in BXH-2 acute myeloid leukemia. We also show that CalDAG-GEF I encodes two protein isoforms, a full-length isoform (CalDAG-GEF Ia) and a C-terminally truncated isoform (CalDAG-GEF Ib). Expression of the full-length CalDAG-GEF Ia isoform in Rat2 fibroblasts enhances growth in low serum, whereas expression in Swiss 3T3 cells causes morphological transformation and increased saturation density. In FDCP1 myeloid cells, CalDAG-GEF Ia expression increases growth and saturation density in the presence of the diacylglycerol analogs phorbol 12-myristate 13-acetate (PMA), which activates CalDAG-GEF Ia exchange activity. Likewise, in 32Dcl3 myeloblast cells, CalDAG-GEF Ia expression increases cell adherence to fibronectin in response to PMA and calcium ionophore and allows higher saturation densities and prolonged growth on fibronectin-coated plates. These effects were correlated with increased Rap1, but not Ras, protein activation following PMA and calcium ionophore treatment. Our results suggest that Rap1-GTP delivers signals that favor progression through the cell cycle and morphological transformation. The identification of CalDAG-GEF I as a proto-oncogene in BXH-2 acute myeloid leukemia is the first evidence implicating Rap1 signaling in myeloid leukemia.