Independent Cell Cultures Research Articles

Importin KPNA2 Is Required for Proper Nuclear Localization and Multiple Functions of NBS1

Nijmegen breakage syndrome (NBS) is a chromosomal-instability syndrome associated with cancer predisposition, radiosensitivity, microcephaly, and growth retardation. The NBS gene product, NBS1, is a component of the MRE11-RAD50-NBS1 (MRN) complex, a central player associated with double strand break (DSB) repair. In response to radiation, NBS1 is phosphorylated by ATM, and the MRN complex relocalizes to form punctate nuclear foci for DNA repair. NBS1 controls both the nuclear localization of the MRN complexes and radiation-induced focus formation. We report here that the KPNA2 (importin alpha1) is important for the normal nuclear localization of the MRN complex and its proper formation of the nuclear foci. KPNA2 is the only member of the importin alpha family that physically interacts with NBS1, and the KPNA2-mediated nucleus localization sequence (NLS) is mapped to amino acid residues 461-467 of NBS1 that is sufficient for both the interaction with KPNA2 and the proper nuclear localization. Inhibition of KPNA2 or blockage of the KPNA2 interaction with NBS1 results in a reduction of radiation-induced nuclear focus accumulation, DSB repair, and cell cycle checkpoint signaling of NBS1. Collectively, our results strongly suggest that an interaction with KPNA2 contributes to nuclear localization and multiple tumor suppression functions of the NBS1 complex.

Identification of Dynamic Proteome Changes Upon Ligand Activation of Trk-Receptors Using Two-dimensional Fluorescence Difference Gel Electrophoresis and Mass Spectrometry

The TrkA and TrkB tyrosine kinases are members of the neurotrophin receptor family and mediate survival, differentiation, growth, and apoptosis of neurons in response to stimulation by their ligands, NGF and BDNF, respectively. Expression levels of TrkA/TrkB are important prognostic factors in a variety of embryonal tumors including neuroblastoma, the most common solid tumor of childhood. Because TrkA/TrkB exhibit a high level of sequence similarity and use overlapping pathways for signal transduction, the existence of specific effector molecules crucial for receptor and cell-type-specific response is likely. To identify these effectors by analyzing biological effects of TrkA and TrkB activation in a defined model, we performed a proteome study using the human neuroblastoma SY5Y cell line stably transfected with the TrkA or TrkB cDNA. The use of the recently introduced DIGE (fluorescence two-dimensional difference gel electrophoresis) system (Amersham Biosciences, Piscataway, NJ) allowed us to monitor differences in protein expression between samples in one gel. Proteomic changes were monitored in a time course of 0, 0.5, 1, 6, and 24 h following receptor activation. Using MALDI mass spectrometry, we identified, respectively, 22 and 9 differentially expressed proteins upon the addition of neurotrophin in SY5Y-TrkB and SY5Y-TrkA cells. Functional assignment revealed that the majority of these proteins are involved in organization and maintenance of cellular structures.

580 Prostate cancer apoptosis in androgen dependent and independent cell cultures harvested from in vivo specimen

580 Prostate cancer apoptosis in androgen dependent and independent cell cultures harvested from in vivo specimen

Mosaic (MSC) cucumbers regenerated from independent cell cultures possess different mitochondrial rearrangements.

Passage of the highly inbred cucumber ( Cucumis sativus L.) line B through cell culture produces progenies with paternally transmitted, mosaic (MSC) phenotypes. Because the mitochondrial genome of cucumber shows paternal transmission, we evaluated for structural polymorphisms by hybridizing cosmids spanning the entire mitochondrial genome of Arabidopsis thaliana L. to DNA-gel blots of four independently generated MSC and four wild-type cucumbers. Polymorphisms were identified by cosmids carrying rrn18, nad5-exon2, rpl5, and the previously described JLV5 deletion. Polymorphisms revealed by rrn18 and nad5-exon2 were due to one rearrangement bringing together these two coding regions. The polymorphism revealed by rpl5 was unique to MSC16 and was due to rearrangement(s) placing the rpl5 region next to the forward junction of the JLV5 deletion. The rearrangement near rpl5 existed as a sublimon in wild-type inbred B, but was not detected in the cultivar Calypso. Although RNA-gel blots revealed reduced transcription of rpl5 in MSC16 relative to wild-type cucumber, Western analyses revealed no differences for the RPL5 protein and the genetic basis of the MSC16 phenotype remains enigmatic. We evaluated 17 MSC and wild-type lines regenerated from independent cell-culture experiments for these structural polymorphisms and identified eight different patterns, indicating that the passage of cucumber through cell culture may be a unique mechanism to induce or select for novel rearrangements affecting mitochondrial gene expression.

Identification of differential gene expression profiles in rat cortical cells exposed to the neuroactive agents trimethylolpropane phosphate and bicuculline

Recent technological advancements in microfabrication combined with the rapid acquisition of full genome sequence data have led to the development of DNA arrays that have the capacity to monitor the expression levels of thousands of genes simultaneously. The development of this technology enables the use of functional genomics approaches to identify molecular markers associated with cellular responsiveness to cytotoxic exposures. Databases containing unique cell-response profiles associated with specific toxicants or classes of toxicants can then be used in conjunction with cell-based biosensor platforms for environmental surveillance and toxicological assessment. An important issue that must be addressed, however, is whether DNA arrays can be used to identify transient gene modulation events in a reproducible manner. To address this issue, we utilized a primary embryonic rat (day 18) cortical cell model system and examined the RNA of both chemically treated and untreated cells using radioisotope-labeled cDNA probes and commercially available nylon membrane arrays. Using this approach, we examined experimental variability, basal gene expression variability, the occurrence of false positives, and the reproducibility of gene expression profiles obtained after chemical exposure. Minimal differences in gene modulation were observed between RNA samples from independently cultured cortical cells when array experiments were conducted in parallel (Pearson correlation coefficient for gene intensities =0.98). In contrast, significant differences in gene expression were observed between array experiments conducted at different times with an identical RNA source (Pearson correlation coefficient for gene intensities=0.91). Our results suggest the effect of basal gene activity differences in independently isolated cell cultures is negligible and that experimental variability possibly associated with the handling of RNA samples, differences in reverse transcription efficiency, hybridization, and/or signal acquisition are the primary contributors to variability in measurements. Using cDNA array analysis of unexposed cells from three independent cell culture preparations, we calculated false positive gene modulation events as a function of the threshold ∣log 2 R∣>1.0. The number of false positives using this criteria was 1–10 gene/ESTs/5109 actively transcribed gene/ESTs represented on the array. Using three independent replicate experiments of untreated cortical cell cultures, we determined that a threshold criterion of ∣log 2 R∣>0.63 for triplicate experiments would reduce the expected number of false positives in our experiments to less than one. Using this criterion, reproducible gene expression profiles were identified in cortical cells exposed to the neuroactive agents trimethylolpropane phosphate and bicuculline.

Development and application of computer software for cell culture laboratory management.

Prototype computer software for a Cell Culture Laboratory Management System (CCLMS) has been developed to relieve cell culture specialists of the burden of manual recordkeeping. Conventional data archives in cell culture laboratories are prone to error and expensive to maintain. The reliance upon cell culture to provide models for biochemical and molecular biological research serves to magnify errors at great expense. The CCLMS prototype encapsulates a modular software application that manages the many aspects of cell culture laboratory recordkeeping. A transaction-based database stores detailed information on subcultures, freezes and thaws, prints waterproof labels for culture vessels, and provides for immediate historical trace-back of any cultured cell line. Linked database files store information specific to an individual culture flask while removing redundancy between similar groups of flasks. A frozen cell log maintains locations of all vials within any type of cryogenic storage unit, locates spaces for newly frozen cell lines, and generates alphabetical or numerical reports. Finally, modules for maintaining cell counts, user records, and culture vessel specifications to support a comprehensive automation process are incorporated within this software. The developed CCLMS prototype has been demonstrated to be an adaptable, reliable tool for improving training, efficiency, and historical rigor for two independent cell culture facilities.

Expression of Mutated Poliovirus Receptors in Human Neuroblastoma Cells Persistently Infected with Poliovirus

Poliovirus (PV) is able to establish persistent infections in human neuroblastoma IMR-32 cells [Colbère-Garapin et al. (1989) Proc. Natl. Acad. Sci. USA 86, 7590]. During persistent infection, PV mutants are selected that display substitutions of residues in regions of the capsid known to interact with the PV receptor (PVR), a glycoprotein of the immunoglobulin superfamily. The mechanism of persistent infection in IMR-32 cells may therefore involve the selection of mutant PVRs. To test this hypothesis, the sequences of the PVR mRNAs in uninfected IMR-32 cells and in two independent IMR-32 cell cultures persistently infected with the Mahoney strain of PV type 1 (PV1/Mahoney) were determined. The PVR mRNA population of uninfected cells was homogeneous, and no mutation was repeatedly found, whereas that of persistently infected cells displayed missense mutations. Particular mutations were repeatedly detected, and all of them mapped to the N-terminal domain of PVR (domain 1), which interacts directly with PV. These mutations generated several types of PVR variants with the following substitutions: Ala67→Thr alone, Ala67→Thr associated with Gly39→Ser, and Arg104→Gln. Functional analysis of PVR in murine LM cells, stably expressing each of the PVR forms, showed that the PVR forms selected during persistent infection conferred on LM cells partial resistance to PV1/Mahoney-induced lysis. Although adsorption onto PVR seemed to be independent of the PVR form, an analysis of the conformational changes of the capsid during the early steps of the PV cycle provided evidence that the Ser39/Thr67 and Gln104 substitutions almost halved the conversion of 160S infectious particles into 135S A particles associated with the PV–PVR interaction. Altogether, these findings indicate that during persistent infection, specific mutations were selected in the domain 1 of PVR and that these mutations increased the resistance of cells to PV-induced lysis. These results are discussed in view of the position of the mutations on PVR.

Cultured cell sublines highly susceptible to prion infection.

Cultured cell lines infected with prions produce an abnormal isoform of the prion protein (PrP(Sc)). In order to derive cell lines producing sufficient quantities of PrP(Sc) for most studies, it has been necessary to subclone infected cultures and select the subclones producing the largest amounts of PrP(Sc). Since postinfection cloning can introduce differences between infected and uninfected cell lines, we sought an approach to generate prion-infected cell lines that would avoid clonal artifacts. Using an improved cell blot technique, which permits sensitive and rapid comparison of PrP(Sc) levels in multiple independent cell cultures, we discovered marked heterogeneity with regard to prion susceptibility in tumor cell sublines. We exploited this heterogeneity to derive sublines which are highly susceptible to prion infection and used these cells to generate prion-infected lines without further subcloning. These infected sublines can be compared to the cognate uninfected cultures without interference from cloning artifacts. We also used susceptible cell lines and our modified cell blot procedure to develop a sensitive and reproducible quantitative cell culture bioassay for prions. We found that the sublines were at least 100-fold more susceptible to strain RML prions than to strain ME7 prions. Comparisons between scrapie-susceptible and -resistant cell lines may reveal factors that modulate prion propagation.

Polyclonal expansion of cells with trisomy 7 in synovia from patients with osteoarthritis

Trisomy 7 as the single chromosome aberration has been found in a variety of neoplasms and in normal tissue in the proximity of tumors, as well as in non-neoplastic lesions. Recently, we described a nonrandom pattern of chromosome aberrations, in particular, a gain of chromosome 7, in synovia, cartilage, and osteophytes from patients with osteoarthritis. To study the clonal origin of trisomy 7 in osteoarthritis, multiple synovial samples were collected from five women, all of whom were informative heterozygotes with regard to the X-linked human androgen receptor gene (AR). From each case, three to four independent cell cultures were initiated. Trisomic cell populations were subcloned from the individual cultures, and it was established whether or not the same allele of AR was inactivated in trisomic cells from different parts of the same joint. The finding of a polyclonal X-inactivation pattern in two of the cases provides strong evidence that gain of an extra copy of chromosome 7 occurs independently in multiple cells.

Loss of Functional Beta2-Microglobulin in Metastatic Melanomas From Five Patients Receiving Immunotherapy

In a subset of patients with metastatic melanoma, T lymphocytes bearing the cell-surface marker CD8 (CD8+ T cells) can cause the regression of even large tumors. These antitumor CD8+ T cells recognize peptide antigens presented on the surface of tumor cells by major histocompatibility complex (MHC) class I molecules. The MHC class I molecule is a heterodimer composed of an integral membrane glycoprotein designated the alpha chain and a noncovalently associated, soluble protein called beta2-microglobulin (beta 2m). Loss of beta 2m generally eliminates antigen recognition by antitumor CD8+ T cells. We studied the loss of beta 2m as a potential means of tumor escape from immune recognition in a cohort of patients receiving immunotherapy. We successfully grew 13 independent tumor cell cultures from tumor specimens obtained from 13 patients in a cohort of 40 consecutive patients undergoing immunotherapy for metastatic melanoma and for whom tumor specimens were available. These cell lines, as well as another melanoma cell line (called 1074mel) that had been derived from tumor obtained from a patient in a cytokine-gene therapy study, were characterized in vitro cytofluorometrically for MHC class I expression and by northern and western blot analyses for messenger RNA (mRNA) and protein expression, respectively, and ex vivo by immunohistochemistry. After one melanoma cell line (1074mel) was found not to express functional beta 2m by cytofluorometric analysis, four (31%) of the 13 newly established melanoma cell lines were found to have an absolute lack of functional MHC class I expression. Northern blot analysis of RNA extracted from the five cell lines exhibiting no functional MHC class I expression showed that these cells contained normal levels of alpha-chain mRNA but variable levels of beta 2m mRNA. In addition, no immunoreactive beta 2m protein was detected by western blot analysis. When human beta 2m was transiently expressed with the use of a recombinant vaccinia virus, cell-surface MHC class I expression was reconstituted and the ability of these five cell lines to present endogenous antigens was restored. Immunohistochemical staining of tumor sections revealed a lack of immunoreactive MHC class I in vivo, supporting the notion that the in vitro observations were not artifactual. Furthermore, archival tumor sections obtained from patients prior to immunotherapy were available from three patients and were found to be beta 2m positive. This result was consistent with the hypothesis that loss of beta 2m resulted from immunotherapy. These data suggest that the loss of beta 2m may be a mechanism whereby tumor cells can acquire immunoresistance. This study represents the first characterization of a molecular route of escape of tumors from immune recognition in a cohort of patients being treated with immunotherapy.

Erythropoietic activity in culture media conditioned by rat mesangial cells.

Using a tissue culture technique we examined erythropoietin (EPO) producing cells in rat glomeruli. In 5 out of 6 independent glomerular cell cultures, EPO activity was found in the mesangial-cell proliferating phase, but not in the epithelial-cell proliferating phase. Therefore, mesangial cells seemed to be EPO producing cells.

A colorimetric assay for serial determination of adenosine deaminase activity in small lymphocyte populations

We describe here an easy, sensitive and quantitative assay for the determination of adenosine deaminase activity in small populations of lymphoid cells. Determinations of enzyme activity and protein concentration are based on colorimetric reactions. All steps are directly performed in 96-well flat-bottom microtiter plates: growth of lymphoid cells (1–2 × 10 5 cells needed), detergent-mediated lysis, incubations for enzymatic assay, and reading of colorimetric reactions with an automatic multiscan spectrophotometer. Thus, screening of several hundred independent cell cultures is easily done within a day. Applications to the detection of rare adenosine deaminase variants from large cell populations and to routine medical assays are considered.

Somatic cell hybrids producing antibodies specific for the tumor antigen of simian virus 40.

We have produced somatic cell hybrids between mouse myeloma cells deficient in hypoxanthine phosphoribosyltransferase IMP: pyrophosphate phosphoribosyltransferase; EC 2.4.2.8) and spleen cells derived from mice primed with either syngeneic or allogeneic cells transformed by simian virus 40. Such hybrids produced antibodies specific for simian virus 40 tumor (T) antigen. Only four of twelve independent hybrid cell cultures produced antibodies against simian virus 40 T antigen that crossreacted with the T antigen induced by BK virus, a human papovavirus isolated from patients who had undergone immunosuppressive therapy.