Infusion of Some but Not All Types of Human Perinatal Stromal Cells Prevent Organ Fibrosis in a Humanized Graft versus Host Disease Murine Model.

Allogeneic transplant rejection represents a medical complication that leads to high morbidity and mortality rates. There are no treatments to effectively prevent fibrosis; however, there is great interest in evaluating the use of perinatal mesenchymal stromal cells (MSCs) and other MSCs to prevent fibrosis associated with chronic rejection. In this study, we isolated human perinatal stromal cells (PSCs) from amnion (AM-PSC), placental villi (PV-PSC), and umbilical cord (UC-PSC) tissues, demonstrating the phenotypic characteristics of MSCs as well as a >70% expression of the immunomodulatory markers CD273 and CD210. The administration of a single dose (250,000 cells) of each type of PSC in a humanized graft versus host disease (hGvHD) NSG® murine model delayed the progression of the disease as displayed by weight loss and GvHD scores ranging at various levels without affecting the hCD3+ population. However, only PV-PSCs demonstrated an increased survival rate of 50% at the end of the study. Furthermore, a histopathological evaluation showed that only PV-PSC cells could reduce human CD45+ cell infiltration and the fibrosis of the lungs and liver. These findings indicate that not all PSCs have similar therapeutic potential, and that PV-PSC as a cell therapeutic may have an advantage for targeting fibrosis related to allograft rejection.

Therapeutic Use of Estrogen Receptor ß Agonists in Prevention and Treatment of Endocrine Therapy Resistant Breast Cancers: Observations From Preclinical Models.

Breast cancer is one of the most common cancers in the world. The majority of breast cancers express estrogen receptor (ER)α, and endocrine therapy is the primary therapeutic approach to treat ER positive breast cancers. However, developing resistance and side effects are common events of these therapeutic strategies. Recent studies have evaluated the role of ERs sub types and demonstrated that ERα is a tumorigenic and ERß functions as a tumor suppressor. In recent years, preclinical studies focused on the use of natural and synthetic ERß agonists to treat wide varieties of cancers, including breast cancer. Successful studies conducted so far, have established that ERß agonists are effective both alone and in combination with chemotherapeutic agents. These data have suggested that use of ERß agonists in combination with endocrine therapy may be an effective treatment strategy in hormone receptor positive breast cancers. The present review focuses on the tumor suppressive role of ERß, and the efficacy and mechanisms of several natural and synthetic ERß agonists against breast cancer.

Clinical and Pathologic Features of Hispanic Endometrial Cancer Patients With Loss of Mismatch Repair Expression.

OBJECTIVES: Approximately 3% to 5% of endometrial cancers (EC) are associated with Lynch syndrome (LS). The clinical characteristics and prevalence of LS have not been well studied in the US Hispanic population. Hispanics are the largest and fastest growing ethnic minority group in the United States. We sought to characterize the demographics, tumor characteristics, and prevalence of loss of mismatch repair (MMR) protein expression in a large Hispanic population with EC. METHODS: From January 1, 2005, to August 1, 2012, 83 women of Hispanic ethnicity diagnosed with EC 50 years and younger were identified. Clinical and pathologic data were abstracted from the electronic medical record. Tumor studies included immunohistochemistry of MLH1, MSH2, MSH6, and PMS2 and methylation of the MLH1 promoter. RESULTS: Ninety-five percent of patients were overweight or obese. The mean body mass index was 40.1 kg/m, 75% had irregular menses, 36% had diabetes, 46% were nulliparous, and 95% had endometrioid histology. Thirteen patients (15.7%) had tumor MMR deficiency due to a presumed germline mutation (9 MSH6, 3 MSH2, and 1 MLH1). The pattern of MMR protein loss was consistent with the expected binding properties of the MMR heterodimer complexes. No significant difference was found in clinical or pathological variables between patients with and without MMR deficient tumors. CONCLUSIONS: The prevalence of molecular findings consistent with LS was at least as high as other populations of varied geography, race, and ethnicity. We found no reliable factors to include body mass index, family history, synchronous tumors, or pathologic tumor features to serve as triage markers for which ECs should be screened for MMR protein loss. Our findings support a recommendation for universal screening of ECs utilizing 2-antibody testing with MLH1 promoter methylation testing as indicated up to 60 years or older. Our recommendations should be generalizable to other Hispanic populations in the Southern United States.

Effects of Combination of Estradiol with Selective Progesterone Receptor Modulators (SPRMs) on Human Breast Cancer Cells In Vitro and In Vivo.

Use of estrogen or estrogen/progestin combination was an approved regimen for menopausal hormonal therapy (MHT). However, more recent patient-centered studies revealed an increase in the incidence of breast cancer in women receiving menopausal hormone therapy with estrogen plus progestin rather than estrogen alone. Tissue selective estrogen complex (TSEC) has been proposed to eliminate the progesterone component of MHT with supporting evidences. Based on our previous studies it is evident that SPRMs have a safer profile on endometrium in preventing unopposed estrogenicity. We hypothesized that a combination of estradiol (E2) with selective progesterone receptor modulator (SPRM) to exert a safer profile on endometrium will also reduce mammary gland proliferation and could be used to prevent breast cancer when used in MHT. In order to test our hypothesis, we compared the estradiol alone or in combination with our novel SPRMs, EC312 and EC313. The compounds were effectively controlled E2 mediated cell proliferation and induced apoptosis in T47D breast cancer cells. The observed effects were found comparable that of BZD in vitro. The effects of SPRMs were confirmed by receptor binding studies as well as gene and protein expression studies. Proliferation markers were found downregulated with EC312/313 treatment in vitro and reduced E2 induced mammary gland proliferation, evidenced as reduced ductal branching and terminal end bud growth in vivo. These data supporting our hypothesis that E2+EC312/EC313 blocked the estrogen action may provide basic rationale to further test the clinical efficacy of SPRMs to prevent breast cancer incidence in postmenopausal women undergoing MHT.

Impaired Development of Early Endometriotic Lesions in CD44 Knockout Mice.

Previous studies have shown endometrial cell (EC) CD44 and peritoneal mesothelial cell (PMC)-associated hyaluronan (hyaluronic acid [HA]) are involved in the attachment of endometrial stroma and epithelial cells to peritoneal mesothelium. Here we assess the CD44-HA interaction in the formation of the early endometriotic lesion using CD44(-/-) (knockout) mice. Using an established murine model and crossover technique, endometrial tissue from donor mice (wild type [WT] and CD44(-/-)) was used to induce endometriosis in recipient mice (WT and CD44(-/-)). Endometriotic lesions were visualized by fluorescent microscopy and confirmed by hematoxylin and eosin staining. Early endometriotic lesions were decreased when CD44(-/-) endometrium was placed in WT recipients and when WT endometrium was placed in CD44(-/-) recipients (P = .002). Early endometriotic lesions were also significantly decreased when both peritoneal and endometrial tissues lacked CD44 expression (P < .01). These studies demonstrate that both EC and PMC CD44 play a role in the development of early endometriotic lesion.

Differentiated adipose-derived stem cell cocultures for bone regeneration in polymer scaffolds in vivo.

Critical-sized bone defects can lead to significant morbidity, and interventions are limited by the availability and donor-site morbidity of bone grafts. Polymer scaffolds seeded with cells have been explored to replace bone grafts. Adipose-derived stem cells have shown great promise for vascularization and osteogenesis of these constructs, and cocultures of differentiated stem cells are being explored to augment vessel and bone formation. Adipose-derived stem cells were differentiated into endothelial cells and osteoblasts, and in vitro studies showed increased proliferation of cocultured cells compared with undifferentiated adipose-derived stem cells and monocultures of endothelial cells and osteoblasts. The cells were seeded into polylactic acid gas-plasma-treated scaffolds as cocultures and monocultures and then implanted into critical-sized rat calvarial defects. The cocultures were in a 1:1 osteoblast to endothelial cell ratio. The increase in proliferation seen by the cocultured cells in vitro did not translate to increased vascularization and osteogenesis in vivo. In vivo, there were trends of increased vascularization in the endothelial cell group and increased osteogenesis in the osteoblast and endothelial monoculture groups, but no increase was seen in the coculture group compared with the undifferentiated adipose-derived stem cells. Endothelial cells enhance vascularization and osteoblast and endothelial cell monocultures enhance bone formation in the polymer scaffold. Predifferentiation of adipose-derived stem cells is promising for improving vascularization and osteogenesis in polymer scaffolds but requires future evaluation of coculture ratios to fully characterize this response.

Gossypin as a novel selective dual inhibitor of V-RAF murine sarcoma viral oncogene homolog B1 and cyclin-dependent kinase 4 for melanoma.

Mutation in the BRAF gene (BRAFV600E) exists in nearly 70% of human melanomas. Targeted therapy against BRAFV600E kinase using a recently identified RAF-selective inhibitor, PLX4032, has been successful in early clinical trials. However, in patients with the normal BRAF allele (wild-type), PLX4032 is protumorigenic. This conundrum identifies the unmet need for novel therapeutic agents to target BRAFV600E kinase that are not counterproductive. We have identified gossypin, a pentahydroxy flavone, as a potent antimelanoma agent. Gossypin inhibited human melanoma cell proliferation, in vitro, in melanoma cell lines that harbor both BRAFV600E kinase and cyclin-dependent kinase 4 (CDK4) as well as in cells with BRAF wild-type allele. Gossypin inhibited kinase activities of BRAFV600E and CDK4, in vitro, possibly through direct binding of gossypin with these kinases, as confirmed by molecular docking studies. For cells harboring the BRAFV600E, gossypin inhibited cell proliferation through abrogation of the MEK-ERK-cyclin D1 pathway and in cells with BRAF wild-type allele, through attenuation of the retinoblastoma-cyclin D1 pathway. Furthermore, gossypin significantly inhibited melanoma growth in an organotypic three-dimensional skin culture mimicking human skin. Gossypin (10 and 100 mg/kg) treatment for 10 days in human melanoma (A375) cell xenograft tumors harboring BRAFV600E significantly reduced tumor volume through induction of apoptosis and increased survival rate in mice, and the effect was significantly superior to that of PLX4032 (10 mg/kg) or roscovitine 10 mg/kg. In summary, this study identified gossypin as a novel agent with dual inhibitory effects for BRAFV600E kinase and CDK4 for treatment of melanoma.

Effect of endothelial differentiated adipose-derived stem cells on vascularity and osteogenesis in poly(D,L-lactide) scaffolds in vivo.

Prevascularization of engineered bony constructs can potentially improve in vivo viability. However, the effect of endothelial cells on osteogenesis is unknown when placed in poly(D,L-lactide) (PLA) scaffolds alone. Adipose-derived stem cells (ASCs) have the ability to differentiate into both osteoblasts and endothelial cells by culture in specific media. We hypothesized that ASC-derived endothelial cells would improve vascularity with minimal contribution to bone formation when placed in scaffold alone. ASCs were successfully differentiated into endothelial cells (ASC-Endo) and osteoblasts (ASC-Osteo) using media supplemented with vascular endothelial growth factor and bone morphogenic protein 2, respectively. Tissue-engineered constructs were created with PLA matrices containing no cells (control), undifferentiated ASCs (ASCs), osteogenic-differentiated ASCs (ASC-Osteo), or endothelial differentiated ASCs (ASC-Endo), and these constructs were evaluated in critical-size Lewis rat calvarial defect model (n = 34). Eight weeks after implantation, the bone volume and microvessel population of bony constructs were evaluated by micro-computed tomography analysis and histologic staining. Bone volumes for ASCs and ASC-Osteo constructs, 0.7 and 0.91 mm(3), respectively, were statistically greater than that for ASC-Endo, 0.28 mm(3) (P < 0.05). There was no statistical difference between the PLA control (0.5 mm(3)) and ASC-Endo (0.28 mm(3)) constructs in bone formation. The percent area of microvessels within constructs was highest in the ASC-Endo group, although it did not reach statistical significance (0.065). Prevascularization of PLA scaffold with ASC-Endo cells will not increase bone formation by itself but may be used as a cell source for improving vascularization and potentially improving existing osteoblast function.

Raf-1, a potential therapeutic target, mediates early steps in endometriosis lesion development by endometrial epithelial and stromal cells.

Endometriosis is a hormone-sensitive gynecological disorder characterized by the benign growth of endometrial-like tissue in the pelvic cavity. Endometriotic lesions composed of endometrial stromal cells (ESC) and glandular epithelial cells (EEC) are thought to arise from menstrual endometrial tissue reaching the pelvic cavity via retrograde menstruation. The cause of endometriotic lesion formation is still not clear. Recent evidence suggest that cytokines may play a role in the early development of endometriosis lesions. Because cytokines and growth factors signal via the v-raf-1 murine leukemia viral oncogene homolog 1 (Raf-1) kinase pathway, we have examined the role of Raf-1 in early steps of endometriosis lesion formation, specifically attachment of endometrial cells to peritoneal mesothelial cells (PMC) and invasion of endometrial cells through PMC (trans-mesothelial invasion). Raf-1 antagonist GW5074 decreased attachment to PMC and trans-mesothelial invasion by primary EEC and ESC. Raf-1 also mediated TGFß-induced trans-mesothelial invasion by the established, low-invasive EEC line EM42. TGFß treatment of EEC resulted in Raf-1 phosphorylation at S338 and phosphorylation of ERK, suggesting that TGFß activates Raf-1 signaling in these cells. GW5074 had little effect on ESC proliferation but inhibited EEC growth significantly under reduced serum conditions. Antagonizing Raf-1 activity and expression via GW5074 and specific Raf-1 small interfering RNA, respectively, did not alter EEC resistance to growth inhibition by TGFß. Raf-1 inhibition blocked induction of EEC growth by epidermal growth factor. Our data suggest that Raf-1 may mediate pathologic steps involved in early endometriosis lesion formation and may be a mediator of TGFß and epidermal growth factor actions in endometriosis.

Effect of adipose tissue-derived osteogenic and endothelial cells on bone allograft osteogenesis and vascularization in critical-sized calvarial defects.

The use of processed bone allograft to repair large osseous defects of the skull has been limited, given that it lacks the osteogenic cellularity and intrinsic vascular supply which are essential elements for successful graft healing and, at the same time, the areas to be targeted through tissue-engineering applications. In this study, we investigated the effect of predifferentiated rat adipose tissue-derived osteoblastic cells (OBs) and endothelial cells (ECs) on calvarial bone allograft healing and vascularization using an orthotopic critical-sized calvarial defect model. For this purpose, thirty-seven 8 mm critical calvarial defects in Lewis rats were treated with bone allografts seeded with no cells, undifferentiated adipose tissue-derived stem cells (ASC), OBs, ECs, and OBs and ECs simultaneously. After 8 weeks, the bone volume and mineral density were calculated using microcomputed tomography and the microvessel formation using immunohistochemical staining and imaging software. The amount of bone within the 8 mm defect was significantly higher for the allografts treated with ECs compared with the allografts treated with OBs (p=0.05) and simultaneously with the two cell lineages (p=0.02). There were no significant differences in bone formation between the latter two groups and the control groups (allografts treated with no cells and undifferentiated ASC). There were no significant differences in bone mineral density among the groups. The amount of microvessels was significantly higher in the group treated with ECs relative to all groups (p=< 0.05). Our results show that the implantation of ASC-derived ECs improves the vascularization of calvarial bone allografts at 8 weeks after treatment. This cell-based vascularization strategy can be used to improve the paucity of perfusion in allogenic bone implants. However, in this study, the treatment of allografts with OBs alone or in combination with ECs did not support bone formation or vascularization.

Estrogen receptor-beta mediates the protective effects of aromatase induction in the MMTV-Her-2/neu x aromatase double transgenic mice.

Breast cancers amplified for the tyrosine kinase receptor Her-2/neu constitute ~30% of advanced breast cancer cases, and are characterized by hormone independence and aggressive growth, implicating this pathway in breast oncogenesis. The induction of Her-2/neu leads to tumor development in 60% of transgenic mice. We have previously examined the effects of estrogen in the MMTV-Her-2/neu background by generating the MMTV-Her-2/neu x aromatase double transgenic mouse strain. MMTV-Her-2/neu x aromatase mice developed fewer mammary tumors than the Her-2/neu parental strain. Our present data show the induction of several estrogen-related genes, including the tumor suppressors BRCA1 and p53, and a decrease in several angiogenic factors. The phosphorylated forms of MAPK p42/44 and AKT were lower in the MMTV-Her-2/neu x aromatase double transgenic mice compared to the MMTV-Her-2/neu parental strain; conversely, phospho-p38 levels were higher in the double transgenic strain. The ERß-selective antagonist THC reversed these changes. The regulation of these factors by ERß was confirmed in clones of MCF7 breast cancer cells overexpressing Her-2/neu in combination with ERß, suggesting that ERß may play a direct role in regulating MAPK and AKT pathways. In summary, the data suggest that ERß may play a major role in decreasing tumorigenesis and that it may affect breast cancer cell proliferation and survival by altering MAPK and AKT activation as well as modulation of tumor suppressor and angiogenesis factors. Treatment with selective ERß agonist may provide therapeutic advantages for the treatment and prevention of breast cancer.

Novel function of transcription factor Nrf2 as an inhibitor of RON tyrosine kinase receptor-mediated cancer cell invasion.

Recepteur d' origine nantais (RON), a tyrosine kinase receptor, is aberrantly expressed in human tumors and promotes cancer cell invasion. RON receptor activation is also associated with resistance to tamoxifen treatment in breast cancer cells. Nrf2 is a positive regulator of cytoprotective genes. Using chromatin immunoprecipitation (ChIP) and site-directed mutagenesis studies of the RON promoter, we identified Nrf2 as a negative regulator of RON gene expression. High Nrf2 and low RON expression was observed in normal mammary tissue whereas high RON and low or undetectable expression of Nrf2 was observed in breast tumors. The Nrf2 inducer sulforaphane (SFN) as well as ectopic Nrf2 expression or knock-down of the Nrf2 negative regulator keap1, which stabilizes Nrf2, inhibited RON expression and invasion of carcinoma cells. Consequently, our studies identified a novel functional role for Nrf2 as a "repressor" and RON kinase as a molecular target of SFN, which mediates the anti-tumor effects of SFN. These results are not limited to breast cancer cells since the Nrf2 inducer SFN stabilized Nrf2 and inhibited RON expression in carcinoma cells from various tumor types.

Immunologic parameters during NOTES compared with laparoscopy in a randomized blinded porcine trial.

BACKGROUND: It remains unclear if the natural orifice translumenal endoscopic surgery (NOTES) technique is less invasive than laparoscopy. Serum interleukins and peritoneal cellular response have been utilized to support the immunologic difference between open and laparoscopic surgery. We hypothesized that there would be no difference between cytokine levels during NOTES or laparoscopic peritoneoscopy. METHODS: Twelve pigs were assigned to NOTES or standard laparoscopy with permuted block randomization. Each group underwent 90 min of diagnostic peritoneoscopy using CO(2) for laparoscopy and air for NOTES pneumoperitoneum. Blood draws were obtained at baseline, at procedure end, and on postoperative days (POD) 1, 2, and 7. Quantification of cytokines (IL-1b and TNF-alpha) was performed with a Duo Set Porcine enzyme-linked immunosorbent assay (ELISA). Laboratory results were captured by a technician blinded to the research question, and data analysis was performed by an investigator blinded to the procedure using t-test and repeated measures linear model. The study was approved by the institutional animal care and use committee (IACUC). RESULTS: All procedures were successfully completed. One NOTES animal succumbed to hemorrhagic gastritis (day 3). All other animals thrived to POD 14, with no gross infections at necropsy. Animals undergoing laparoscopy had lower mean arterial pH than NOTES animals (p < 0.001). Serum and intraperitoneal white blood cell (WBC) counts were similar between the groups. Mean interleukin-1b levels at baseline, at the end of the procedure and at 48 h did not differ (0.50 and 0.31; p = 0.65). TNF-alpha levels did not differ at baseline or procedure end but increased in the NOTES group on POD 1, persisting to POD 7. Tumor necrosis factor-alpha (TNF-alpha) decreased in the laparoscopy group (p = 0.005). CONCLUSION: Cytokines and WBC did not differ between laparoscopic and NOTES groups during the initial 24 h. These findings do not currently support the assumption that NOTES is less invasive than laparoscopy. The late TNF-alpha elevation contradicts other studies and requires further examination.

Flavonoid, silibinin, inhibits proliferation and promotes cell-cycle arrest of human colon cancer.

BACKGROUND: Anti-oxidative extracts from the milk thistle plant (Silybum marianum) have been shown to have antiproliferative effects in several tumor types. Silibinin is the primary active component isolated from the crude seed extract, silymarin. It has been used as a dietary supplement for hepatoprotection for over 2000 years. Silibinin has been shown to be safe in multiple animal models and has had no significant adverse events in human studies. We investigated the potential for this nontoxic flavolignan to inhibit proliferation of human colon cancer. MATERIALS AND METHODS: Three well-characterized cell lines, Fet, Geo, and HCT116, were studied. The MTT cell-viability assay was performed to study the effect of silibinin on proliferation. Fluorescence-activated cell sorter (FACS) analysis was used to determine the effects of silibinin on cell cycle and apoptosis. 4', 6'-diamidine-2'-phenylindole (DAPI) staining with confocal microscopy was used to morphologically confirm these results. Poly ADP-ribose polymerase (PARP) cleavage and expression levels of p21, p27, cyclins B1/D1, and CDK-2 were measured. Cyclooxygenase-2 (COX-2) levels were also measured. The experiments were performed in triplicate and reported as mean values with standard errors. Means were contrasted using analysis of variance with Dunnet's correction for multiple testing. All statistical testing was two-sided with a significance level of 5%. RESULTS: The MTT assay revealed a strong dose-dependent inhibitory effect. Treatment with 75 microg/mL resulted in 50% inhibition of cell-viability (IC-50) in Fet and Geo lines at 72 h. An IC50 dose of 40 ug/mL was obtained in HCT116, a poorly-differentiated cell line, at 72 h. FACS analysis demonstrated statistically significant cell-cycle arrest in all cell lines. G(2)-M phase arrests in Fet and Geo cell lines (P < 0.001) and a G1 arrest in HCT116 (P = 0.005) were noted. Trivial increases in early apoptotic rates (2% to 3%) for Geo and HCT116 were noted on FACS analysis via annexin V-propidium iodide technique (P < 0.05), but no evidence for apoptosis was seen on Western blot for PARP cleavage or DAPI. Cyclin B1/D1 and CDK-2 levels were inhibited. Increased expression of cell cycle inhibitors, p21 or p27, was noted, and there was no effect on COX-2 expression. CONCLUSIONS: Silibinin significantly inhibits proliferation through cell-cycle arrest via inhibition of cyclin-CDK promoter activity. Despite its antioxidant profile, there is no effect on COX-2 expression. Apoptosis does not appear to be greatly increased in human colon cancer cell lines Fet, Geo, and HCT116. Rather, inhibition of cell cycle regulatory proteins plays a fundamental role in silibinin's mechanism of action, and this may serve as a basis for combined use with conventional chemotherapeutics.

Determining risk of biochemical recurrence in prostate cancer by immunohistochemical detection of PTEN expression and Akt activation.

PURPOSE: A considerable fraction of patients who undergo radical prostatectomy as treatment for primary prostate cancer experience biochemical recurrence detected by elevated serum levels of prostate-specific antigen. In this study, we investigate whether loss of expression of the tumor suppressor phosphatase and tensin homologue deleted on chromosome 10 (PTEN) and the phosphorylated form of the cell survival protein Akt (pAkt) predicts biochemical recurrence. EXPERIMENTAL DESIGN: Expression of PTEN and pAkt was detected by immunohistochemistry in paraffin-embedded prostate cancer tissue obtained from men undergoing radical prostatectomy. Outcome was determined by 60-month follow-up determining serum prostate-specific antigen levels. RESULTS: By itself, PTEN was not a good predictor of biochemical recurrence; however, in combination with pAkt, it was a better predictor of the risk of biochemical recurrence compared with pAkt alone. Ninety percent of all cases with high pAkt and negative PTEN were recurrent whereas 88.2% of those with low pAkt and positive PTEN were nonrecurrent. In addition, high Gleason scores resulted in reduced protection from decreased pAkt and increased PTEN. By univariate logistic regression, pAkt alone gives an area under the receiver-operator characteristic curve of 0.82 whereas the area under the receiver-operator characteristic curve for the combination of PTEN, pAkt, and Gleason based on a stepwise selection model is 0.89, indicating excellent discrimination. CONCLUSIONS: Our results indicate that loss of PTEN expression, together with increased Akt phosphorylation and Gleason score, is of significant predictive value for determining, at the time of prostatectomy, the risk of biochemical recurrence.

Compensatory increases in Her-2/neu activation in response to EGFR tyrosine kinase inhibition in colon cancer cell lines.

BACKGROUND: Tyrosine kinase receptors of the ErbB family have become promising targets for anti-neoplastic drugs, but mechanisms of resistance are incompletely understood. To investigate such pathways, we applied a small-molecule, selective EGFR inhibitor, OSI-774, to three well-characterized colon cancer cell lines and studied the alterations of expression and activation of receptors in the erbB family. METHODS: MTT assays were performed to determine the IC(50)s of GEO, FET, and HCT 116 human colorectal cancer cell lines treated with OSI-774. Plated cells were then exposed to either DMSO control or 7 microm of OSI-774 for treatment durations of 1, 3, 5, 7, 10, 14, 28, and 56 days. Cell lysates were evaluated by Western blotting, evaluating both total and phosphorylated levels of EGFR, Her-2/neu, and erbB-3. RESULTS: IC(50) values for GEO, FET, and HCT 116 cell lines exposed to OSI-774 were 12.0, 16.0, and greater than 100 microm, respectively. In all treated cell lines, OSI-774 diminished EGFR activation but did not affect total expression compared with controls. In contrast, Her-2/neu activation was increased in all cell lines. These changes in EGFR and Her-2/neu were identified within 24 h but peaked later in the treatment cycle. ErbB-3 expression and activation did not follow a consistent pattern between cell lines. CONCLUSIONS: Inhibition of EGFR led to increased activation of Her-2/neu. This result suggests a possible mechanism by which cells might escape the proapoptotic signals resulting from EGFR blockade. Our findings suggest concurrent inhibition of multiple members of the erbB family may yield stronger apoptotic responses than single receptor blockade alone.

Knockdown of prothrombin in zebrafish.

Thrombin is a serine protease generated from its zymogen, prothrombin, and plays a central role in the coagulation cascade. It is also important for mammalian development. The zebrafish has now been established as an excellent genetic model for studies on mammalian hemostasis and development. In this report, we used prothrombin-specific antisense morpholinos to knock down the levels of prothrombin to characterize the effects of prothrombin deficiency in the zebrafish embryo. Prothrombin morpholino-injected zebrafish embryos yielded an early phenotype exhibiting severe abnormalities that later showed occasional bleeding. In a second late phenotype, the embryos had no observable morphological abnormalities in early stages, but showed occasional bleeding at later stages. These phenotypes resembled characteristics shown by prothrombin knockout mice. Laser-induced vascular injury on some of the normal appearing phenotypic larvae showed a prolonged time to occlusion, and recombinant zebrafish prothrombin injected into these larvae restored a normal time to occlusion thus showing the specificity of the morpholino effect. The system developed here should be useful for investigation of the role of thrombin in vertebrate development.