Cholesterol-Depletion-Induced Membrane Repair Carries a Raft Conformer of P-Glycoprotein to the Cell Surface, Indicating Enhanced Cholesterol Trafficking in MDR Cells, Which Makes Them Resistant to Cholesterol Modifications.

The human P-glycoprotein (P-gp), a transporter responsible for multidrug resistance, is present in the plasma membrane's raft and non-raft domains. One specific conformation of P-gp that binds to the monoclonal antibody UIC2 is primarily associated with raft domains and displays heightened internalization in cells overexpressing P-gp, such as in NIH-3T3 MDR1 cells. Our primary objective was to investigate whether the trafficking of this particular P-gp conformer is dependent on cholesterol levels. Surprisingly, depleting cholesterol using cyclodextrin resulted in an unexpected increase in the proportion of raft-associated P-gp within the cell membrane, as determined by UIC2-reactive P-gp. This increase appears to be a compensatory response to cholesterol loss from the plasma membrane, whereby cholesterol-rich raft micro-domains are delivered to the cell surface through an augmented exocytosis process. Furthermore, this exocytotic event is found to be part of a complex trafficking mechanism involving lysosomal exocytosis, which contributes to membrane repair after cholesterol reduction induced by cyclodextrin treatment. Notably, cells overexpressing P-gp demonstrated higher total cellular cholesterol levels, an increased abundance of stable lysosomes, and more effective membrane repair following cholesterol modifications. These modifications encompassed exocytotic events that involved the transport of P-gp-carrying rafts. Importantly, the enhanced membrane repair capability resulted in a durable phenotype for MDR1 expressing cells, as evidenced by significantly improved viabilities of multidrug-resistant Pgp-overexpressing immortal NIH-3T3 MDR1 and MDCK-MDR1 cells compared to their parents when subjected to cholesterol alterations.

Covalently immobilized glycosaminoglycans enhance megakaryocyte progenitor expansion and platelet release.

Application of umbilical cord blood (UCB) transplantation in adults as a treatment post-chemotherapy is hampered due to delayed platelet recovery. A potential solution suggested is the transfusion of ex vivo expanded megakaryocytes (Mks) from hematopoietic stem cells (HSCs). Alternatively, large-scale production of platelets in vitro has also been attempted with the goal of transfusing them into patients with thrombocytopenia. Glycosaminoglycans (GAGs) have been shown to influence the proliferation and differentiation of HSCs. This study sought to examine the effects of immobilized GAGs on the expansion, apoptosis, and platelet release activity of CD41a+ Mk progenitors in vitro. Freshly isolated HSCs from UCB were cultured in serum-free media supplemented with thrombopoietin on GAG-derivatized chitosan membranes for 17 days. Controls consisted of uncoated and chitosan-coated wells. Wells were demidepopulated at periodic intervals and analyzed by flow cytometry. Heparin and dermatan sulfate surfaces significantly enhanced total cell and Mk cell expansion (p < 0.05) compared to both the controls. The apoptotic Mk fraction was significantly lower on GAG surfaces (p < 0.05) compared to the polystyrene control during the early stages of the culture (days 7 and 11). However, by day 17, the apoptotic Mk fraction was comparable on all surfaces. The cumulative number of platelets generated on dermatan sulfate and heparan sulfate surfaces was significantly higher (p < 0.05) than on both the controls. These results suggest that immobilized GAGs delay Mk apoptosis and thereby enhance Mk expansion and platelet production.

Steroid receptor and growth factor receptor expression in human nonsmall cell lung cancers using cells procured by laser-capture microdissection.

Few biomarkers exist for management of nonsmall cell lung cancers (NSCLC), although estrogen receptor (ERalpha and ERbeta) and EGF receptor (EGFR) expression has been related to clinical outcome. To circumvent problems of cellular heterogeneity in whole tissue, relative gene expression of ERalpha, ERbeta, EGFR, and HER-2 (c-erb-B2) was examined in pure lung carcinoma (LC) cells and normal epithelia by LCM. Cell-specific RNA was isolated and purified for RT-qPCR and microarray. Comparison of NSCLC cells to normal epithelia indicated increased levels of mRNA expression of ERbeta, ERalpha, EGFR, and HER-2 by 31%, 38%, 54%, and 62%, respectively, in LCs. The majority of NSCLC exhibiting low ERalpha and high HER-2 expression were from smokers. Although there was no correlation between ERbeta or EGFR expression and smoking history, there appeared to be an inverse relationship between levels of ERbeta and EGFR mRNAs in normal and neoplastic lung. Additionally, microarray analyses of LCM cells revealed >2,000 genes significantly altered in LC compared with normal epithelia. Herein, differences in NSCLC gene expression and normal lung cells were noted between specimens from gender and smoking groups. Microarray data revealed ERa expression was associated with alterations in <20 genes while ERbeta expression revealed >500 associated genes, suggesting a more prominent role for ERbeta in lung. HER-2 mRNA levels appeared associated with >1,000 genes, while EGFR mRNA levels were associated with far fewer genes. Collectively, results suggest quantitative genomic analyses of pure cell populations allow more accurate interpretation of LC status, which is being correlated with clinical outcome.

Application of porous glycosaminoglycan-based scaffolds for expansion of human cord blood stem cells in perfusion culture.

In vitro expansion of hematopoietic stem cells (HSCs) has been employed to obtain sufficient numbers of stem cells for successful engraftment after HSC transplantation. A three-dimensional perfusion bioreactor system with a heparin-chitosan scaffold was designed and evaluated for its capability to support maintenance and expansion of HSCs. Porous chitosan scaffolds were fabricated by a freeze-drying technique and N-desulfated heparin was covalently immobilized within the scaffolds using carbodiimide chemistry. CD34+ HSCs isolated from umbilical cord blood by immunomagnetic separation were cultured within the porous scaffold in a perfusion bioreactor system. Control cultures were maintained on dishes coated with similar heparin-chitosan films. Oxygen uptake was measured during the culture period. After 7 days of culture, scaffolds were harvested for analysis. Cellular phenotype and HSC characteristics were evaluated via flow cytometry and colony forming unit assays. The results indicate good cell retention and proliferation within the perfused scaffolds. Oxygen consumption in the perfusion bioreactor system increased continuously during the culture, indicating steady cell growth. Cells from the perfused scaffold cultures showed higher percentages of primitive progenitors and exhibited superior colony forming unit performance as compared to cells from static cultures. In addition, perfusion culture at low oxygen (5%) enhanced the expansion of CD34+ cells and colony-forming activity compared to high oxygen (19%) cultures. The results suggest that perfusion culture of cord blood CD34+ cells under bone marrow-like conditions enhances HSC expansion compared to static cultures.

Direct detection of herceptin/trastuzumab binding on breast tissue sections.

The protooncogene product HER-2/neu is the target of the humanized monoclonal antibody trastuzumab (Herceptin). Several tests are used clinically to identify patients with HER-2/neu overexpression based on evaluation by pathologists of gene amplification by fluorescence in situ hybridization or protein expression using immunohistochemistry (IHC). A simple technique has been developed for staining formalin-fixed, paraffin-embedded breast cancer tissue using unmodified Herceptin/trastuzumab as the primary antibody. Results were compared with staining with the commercial kit, HercepTest, as well as with polyclonal anti-HER-2/neu antibodies and with biotinylated trastuzumab. These procedures were tested using four breast cancer microarrays. There were 854 cores that were stained with all four antibodies, representing 325 cases. A standard 4-point scoring system (0-3) was used. A total of 156 cases (48%) were scored as 0 by all the methods used and 31 (9.5%) were positive (3+) by all methods. Of interest, three cases scored negative using polyclonal anti-HER-2/neu antibodies but were positive using unmodified trastuzumab. To clarify this discrepancy, whole sections of tumors were examined with both antibodies using double labeling. There were some tumors that demonstrated a mosaic pattern of staining with neighboring cells or groups of cells stained exclusively with one antibody or the other. These results demonstrate that unmodified humanized or human therapeutic antibodies could be used for preclinical testing or in a clinical laboratory setting for IHC-based selection of patients for treatment, and results of such selection could be different from those obtained using polyclonal antibody-based IHC procedure.

Transcriptional profiling of degraded RNA in cryopreserved and fixed tissue samples obtained at autopsy.

BACKGROUND: Traditional multiplexed gene expression methods require well preserved, intact RNA. Such specimens are difficult to acquire in clinical practice where formalin fixation is the standard procedure for processing tissue. Even when special handling methods are used to obtain frozen tissue, there may be RNA degradation; for example autopsy samples where degradation occurs both pre-mortem and during the interval between death and cryopreservation. Although specimens with partially degraded RNA can be analyzed by qRT-PCR, these analyses can only be done individually or at low levels of multiplexing and are laborious and expensive to run for large numbers of RNA targets. METHODS: We evaluated the ability of the cDNA-mediated Annealing, Selection, extension, and Ligation (DASL) assay to provide highly multiplexed analyses of cryopreserved and formalin fixed, paraffin embedded (FFPE) tissues obtained at autopsy. Each assay provides data on 1536 targets, and can be performed on specimens with RNA fragments as small as 60 bp. RESULTS: The DASL performed accurately and consistently with cryopreserved RNA obtained at autopsy as well as with RNA extracted from formalin-fixed paraffin embedded tissue that had a cryopreserved mirror image specimen with high quality RNA. In FFPE tissue where the cryopreserved mirror image specimen was of low quality the assay performed reproducibly on some but not all specimens. CONCLUSION: The DASL assay provides reproducible results from cryopreserved specimens and many FFPE specimens obtained at autopsy. Gene expression analyses of these specimens may be especially valuable for the study of non-cancer endpoints, where surgical specimens are rarely available.

Development of a stereological method to measure levels of fluoropyrimidine metabolizing enzymes in tumor sections using laser scanning cytometry.

BACKGROUND: The enzymes thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) influence the activities of fluoropyrimidine anticancer drugs. The sensitivity of cancer cells to capecitabine, which is an oral, tumor-selective pre-prodrug of 5-fluorouracil may correlate better to the TP/DPD ratio than to levels of either enzyme alone. Our goal was to develop a quantitative immunofluorescent method for estimating the levels of TP, DPD, and their ratio in archival tumor sections. METHODS: Mouse anti-TP and rat anti-DPD monoclonal antibodies were used for parallel indirect immunofluorescent staining. The fluorescence was measured using a laser scanning cytometer (LSC; CompuCyte, Cambridge, MA) in single cells and in sections prepared from cell lines and a human tumor. The phantom contouring feature of the LSC provided a stereologic approach for collecting the fluorescence intensity data from sections. RESULTS: The relative fluorescence intensities measured in single cells or in sections of the cell lines, using single or double labeling, were similar, supporting the suitability of phantom contouring and two-color staining. Sections of the T-24 and ZR-75-1 cell lines placed on the same slide as the tumor section were used as internal standards for fluorescence measurements. The TP/DPD ratios measured in three cell lines correlated well with the cytotoxicity of 5'-deoxy-5-fluorouridine measured in vitro, indicating that the measurements are related to the biological activity of the drug. CONCLUSIONS: Plotting the data as contour maps of the topologic distribution of fluorescence intensities in tumor sections allows subsequent histopathologic examination, which may reveal features of the tumors leading to high or low ratios of these enzymes. In addition, this method can be used for any drug target/metabolic system where the key components are known and suitable antibodies are available.

Laser Scanning Cytometry for selection of green fluorescent protein transgenic mice using small number of blood cells.

A Laser Scanning Cytometry-based method was developed for identification of transgenic mice expressing green fluorescent protein (GFP) using minute amounts of peripheral blood. The difference between the autofluorescence of cells not expressing GFP and the fluorescence of GFP expressing cells after excitation with Ar-ion laser (wavelength 488 nm) and detection of emitted fluorescent light in the green channel was high enough for unambiguous identification of the GFP expressing mice. The sensitivity of this method was estimated 1:10(4) for detection of rare GFP expressing cells under the conditions used. This sensitivity should be sufficient for many studies on microchimerism. Because of the possibility for relocation of the cells, this method will be particularly useful for characterizing the cells with high GFP expression using other markers of cell phenotype or conventional morphological analysis.

Potential for predicting toxicity and response of fluoropyrimidines in patients.

The efficacy of cancer therapy is compromised by the fact that there are currently no good ways to predict which patients will benefit from treatment. This long standing goal is closer to becoming a reality as more is learned about the molecules that affect the activities of various therapeutic agents. The fluoropyrimidine antimetabolites drugs have been in clinical use for over 4 decades and the cellular proteins important for their activities have been studied in detail. The most important are the major target enzyme, thymidylate synthase (TS) and the rate limiting enzyme in the degradation pathway, dihydropyrimidine dehydrogenase (DPD), equally important for the analogue capecitabine is thymidine phosphorylase (TP), which is rate limiting for activation of this prodrug. A number of assays are available for these enzymes, including enzyme activity measurements. quantitative PCR for RNA expression and immunological methods for protein expression. With each of these methods, more clinical studies are required to validate their clinical usefulness.

Uridine phosphorylase is a potential prognostic factor in patients with oral squamous cell carcinoma.

BACKGROUND: Uridine phosphorylase (UPase) catalyzes the reversible phosphorolysis of uridine to uracil. The expression levels and the enzymatic activity of UPase are reported to be higher in human solid tumors than in adjacent normal tissues. However, to the authors' knowledge the clinical significance of UPase expression as determined by immunohistochemical analysis has not been demonstrated in human malignancies. METHODS: The authors prepared the antibody against UPase, examined the staining of UPase in 72 cases of oral squamous cell carcinoma (SCC) with immunohistochemical analysis using the antibody, and analyzed its relation to clinical and pathologic factors. RESULTS: UPase stained mainly within the invasive edges of tumors and macrophages. UPase-positive staining was observed in 56 of the 72 tumors (77.8%). High staining of UPase (defined as > 50% of cells in a tumor biopsy that are positive for UPase) in primary tumors frequently was associated with the presence of metastasis to lymph nodes (P = 0.007 by the Fischer exact test) and with lower overall survival (P = 0.03 by the log-rank test). CONCLUSIONS: The high rate of UPase staining in biopsies from patients with oral SCC may be a prognostic marker for these individuals.

Expression of uridine and thymidine phosphorylase genes in human breast carcinoma.

Uridine phosphorylase (UPase) and an angiogenic enzyme, thymidine phosphorylase (dThdPase) are involved in degradation of the pyrimidine nucleosides through phosphorolysis. The expression levels of UPase and dThdPase are higher in human solid tumors including breast carcinomas than in normal tissues. To clarify the correlation between the expression levels of UPase and dThdPase genes and the clinicopathological factors, mRNA levels of these enzymes were examined by RT-PCR in 43 breast carcinomas. UPase gene expression was not correlated with dThdPase gene expression (regression coefficient R = 0.032). Although the expression level of the dThdPase gene was correlated with angiogenesis, detected by immunostaining endothelial cells (R = 0.66), that of UPase gene was not (R = 0.044). These results suggest that UPase does not have a strong angiogenic activity. The UPase gene expression levels in tumors of patients who relapsed were significantly higher than in those from patients who did not (p = 0.039). Although the expression levels of neither UPase or dThdPase were associated with age, pT, pN, pM, estrogen or progesterone receptor positivity, the patients with the higher levels of UPase gene expression had worse survival (p = 0.0038) than those with lower levels. In contrast, the expression of dThdPase gene was not related to relapse or survival of these patients with breast carcinoma. Our findings suggest that the expression level of UPase gene may be an independent prognostic marker in human breast carcinoma.