Intracellular Calcium Mobilization in Response to Ion Channel Regulators via a Calcium-Induced Calcium Release Mechanism.

Free intracellular calcium ([Ca2+]i), in addition to being an important second messenger, is a key regulator of many cellular processes including cell membrane potential, proliferation, and apoptosis. In many cases, the mobilization of [Ca2+]i is controlled by intracellular store activation and calcium influx. We have investigated the effect of several ion channel modulators, which have been used to treat a range of human diseases, on [Ca2+]i release, by ratiometric calcium imaging. We show that six such modulators [amiodarone (Ami), dofetilide, furosemide (Fur), minoxidil (Min), loxapine (Lox), and Nicorandil] initiate release of [Ca2+]i in prostate and breast cancer cell lines, PC3 and MCF7, respectively. Whole-cell currents in PC3 cells were inhibited by the compounds tested in patch-clamp experiments in a concentration-dependent manner. In all cases [Ca2+]i was increased by modulator concentrations comparable to those used clinically. The increase in [Ca2+]i in response to Ami, Fur, Lox, and Min was reduced significantly (P < 0.01) when the external calcium was reduced to nM concentration by chelation with EGTA. The data suggest that many ion channel regulators mobilize [Ca2+]i We suggest a mechanism whereby calcium-induced calcium release is implicated; such a mechanism may be important for understanding the action of these compounds.

Match criteria for human cell line authentication: where do we draw the line?

Continuous human cell lines have been used extensively as models for biomedical research. In working with these cell lines, researchers are often unaware of the risk of cross-contamination and other causes of misidentification. To reduce this risk, there is a pressing need to authenticate cell lines, comparing the sample handled in the laboratory to a previously tested sample. The American Type Culture Collection Standards Development Organization Workgroup ASN-0002 has developed a Standard for human cell line authentication, recommending short tandem repeat (STR) profiling for authentication of human cell lines. However, there are known limitations to the technique when applied to cultured samples, including possible genetic drift with passage. In our study, a dataset of 2,279 STR profiles from four cell banks was used to assess the effectiveness of the match criteria recommended within the Standard. Of these 2,279 STR profiles, 1,157 were grouped into sets of related cell lines-duplicate holdings, legitimately related samples or misidentified cell lines. Eight core STR loci plus amelogenin were used to unequivocally authenticate 98% of these related sets. Two simple match algorithms each clearly discriminated between related and unrelated samples, with separation between related samples at ≥80% match and unrelated samples at <50% match. A small degree of overlap was noted at 50-79% match, mostly from cell lines known to display variable STR profiles. These match criteria are recommended as a simple and effective way to interpret results from STR profiling of human cell lines.

Function of mutant and wild-type plexinb1 in prostate cancer cells.

BACKGROUND: Semaphorins act as chemotactic cues for cell movement via their transmembrane receptors, plexins. Somatic missense mutations in the plexinB1 gene coupled with overexpression of the protein frequently occur in prostate tumors, indicating a role for plexinB1 in the pathogenesis of prostate cancer. However, the effect of semaphorin/plexin signaling is highly context dependent and whether plexinB1 acts as an inducer or inhibitor of prostate tumor progression in this context is not known. METHODS: The response of prostate cancer cell lines to plexinB1 activation was assessed in migration, invasion, proliferation and protein phosphorylation assays. Expression was assessed by quantitative RTPCR and immunoblotting. RESULTS: Different prostate cancer cell lines respond to Sema4D (the ligand for plexinB1) in diverse ways. Activation of endogenous plexinB1 enhances migration, invasion and anchorage-independent growth of LNCaP prostate cancer cells via activation of ErbB2 and Akt. In contrast, Sema4D-stimulation decreased the motility and proliferative capacity of PC3 cells. LNCaP has a missense mutation (Thr1697Ala) in the plexinB1 gene while LNCaP-LN3, a derivative of LNCaP, expresses high levels of wild-type plexinB1 only. Sema4D stimulation increases the motility and anchorage independent growth of both cell lines, showing that these responses are not dependent on the presence of the Thr1697Ala form of plexinB1. ErbB2 and plexinB1 are expressed in primary prostate epithelial cells. CONCLUSIONS: PlexinB1 signals via ErbB2 to increase the invasive phenotype of prostate cancer cells. Both wild-type and mutant forms of plexinB1 are potential targets for anti-cancer therapy in prostate tumors that express ErbB2.

Curing metastatic cancer: lessons from testicular germ-cell tumours.

Most metastatic cancers are fatal. More than 80% of patients with metastatic testicular germ-cell tumours (TGCTs), however, can be cured using cisplatin-based combination chemotherapy. Why are TGCTs more sensitive to chemotherapeutics than most other tumour types? Answers to this question could lead to new treatments for metastatic cancers.

Effect of cancer-associated mutations in the PlexinB1 gene.

BACKGROUND: Semaphorins act as chemotactic cues for cell movement via their transmembrane receptors, plexins. Somatic missense mutations in the plexinB1 gene coupled with overexpression of the protein frequently occur in prostate tumours, indicating a role for plexinB1 in the pathogenesis of prostate cancer. RESULTS: Two specific mutations found in prostate cancer enhance RhoD binding and one other mutation results in loss of inhibition of Rac-dependent Pak1 phosphorylation and lamellipodia formation and in impairment of trafficking of plexinB1 to the membrane. None of the three characterised mutations affect PDZRhoGEF binding, RhoA activity, the interaction of plexinB1 with the oncogenes ErbB2 or c-Met or ErbB2 phosphorylation. The mutations have the net effect of increasing cell motility by blocking plexinB1-mediated inhibition of Rac while enhancing the interaction with RhoD, an anti-migratory factor. CONCLUSIONS: PlexinB1 mutations block plexinB1-mediated signalling pathways that inhibit cell motility.

Biochemical relapse following radical prostatectomy and miR-200a levels in prostate cancer.

BACKGROUND: Radical prostatectomy cures the majority of men with clinically localized disease, but up to 30% of men relapse with rising serum PSA levels. Stage, Gleason grade, and pre-operative PSA levels are associated with outcome but do not accurately predict which individuals will relapse. MicroRNA (miRNA) levels are altered in cancer and are associated with progression of disease. The miR-200 family has roles in prostate cancer. METHODS: miR-200a levels were measured in 18 radical prostatectomy samples from men who did not relapse and from 18 who did relapse, matched for stage (all T3), grade, and PSA levels. A pair of cancer and normal prostate cell lines derived from the same radical prostatectomy specimen were transfected with miR-200a to determine the effects on growth, wound healing, and invasion. RESULTS: Comparing the matched samples, 11 of the relapsers contained lower, 2 higher and 5 similar levels to the non-relapsers. Transient transfection of miR-200a significantly reduced cell proliferation in prostate cancer cell lines but did not affect invasiveness. CONCLUSION: miR-200a overexpression reduced prostate cancer cell growth and may have potential, in combination with other markers, in stratifying prostate cancer patients for more intensive monitoring and therapy.

PAK4: a pluripotent kinase that regulates prostate cancer cell adhesion.

Hepatocyte growth factor (HGF) is associated with tumour progression and increases the invasiveness of prostate carcinoma cells. Migration and invasion require coordinated reorganisation of the actin cytoskeleton and regulation of cell-adhesion dynamics. Rho-family GTPases orchestrate both of these cellular processes. p21-activated kinase 4 (PAK4), a specific effector of the Rho GTPase Cdc42, is activated by HGF, and we have previously shown that activated PAK4 induces a loss of both actin stress fibres and focal adhesions. We now report that DU145 human prostate cancer cells with reduced levels of PAK4 expression are unable to successfully migrate in response to HGF, have prominent actin stress fibres, and an increase in the size and number of focal adhesions. Moreover, these cells have a concomitant reduction in cell-adhesion turnover rates. We find that PAK4 is localised at focal adhesions, is immunoprecipitated with paxillin and phosphorylates paxillin on serine 272. Furthermore, we demonstrate that PAK4 can regulate RhoA activity via GEF-H1. Our results suggest that PAK4 is a pluripotent kinase that can regulate both actin cytoskeletal rearrangement and focal-adhesion dynamics.

Check your cultures! A list of cross-contaminated or misidentified cell lines.

Continuous cell lines consist of cultured cells derived from a specific donor and tissue of origin that have acquired the ability to proliferate indefinitely. These cell lines are well-recognized models for the study of health and disease, particularly for cancer. However, there are cautions to be aware of when using continuous cell lines, including the possibility of contamination, in which a foreign cell line or microorganism is introduced without the handler's knowledge. Cross-contamination, in which the contaminant is another cell line, was first recognized in the 1950s but, disturbingly, remains a serious issue today. Many cell lines become cross-contaminated early, so that subsequent experimental work has been performed only on the contaminant, masquerading under a different name. What can be done in response-how can a researcher know if their own cell lines are cross-contaminated? Two practical responses are suggested here. First, it is important to check the literature, looking for previous work on cross-contamination. Some reports may be difficult to find and to make these more accessible, we have compiled a list of known cross-contaminated cell lines. The list currently contains 360 cell lines, drawn from 68 references. Most contaminants arise within the same species, with HeLa still the most frequently encountered (29%, 106/360) among human cell lines, but interspecies contaminants account for a small but substantial minority of cases (9%, 33/360). Second, even if there are no previous publications on cross-contamination for that cell line, it is essential to check the sample itself by performing authentication testing.

Plexin-B1 mutations in prostate cancer.

Semaphorins are a large class of secreted or membrane-associated proteins that act as chemotactic cues for cell movement via their transmembrane receptors, plexins. We hypothesized that the function of the semaphorin signaling pathway in the control of cell migration could be harnessed by cancer cells during invasion and metastasis. We now report 13 somatic missense mutations in the cytoplasmic domain of the Plexin-B1 gene. Mutations were found in 89% (8 of 9) of prostate cancer bone metastases, in 41% (7 of 17) of lymph node metastases, and in 46% (41 of 89) of primary cancers. Forty percent of prostate cancers contained the same mutation. Overexpression of the Plexin-B1 protein was found in the majority of primary tumors. The mutations hinder Rac and R-Ras binding and R-RasGAP activity, resulting in an increase in cell motility, invasion, adhesion, and lamellipodia extension. These results identify a key role for Plexin-B1 and the semaphorin signaling pathway it mediates in prostate cancer.

A PAK4-LIMK1 pathway drives prostate cancer cell migration downstream of HGF.

Hepatocyte growth factor (HGF) is associated with tumour progression and increases the invasiveness of prostate carcinoma cells. Cell migration and invasion requires reorganisation of the actin cytoskeleton; processes mediated by the Rho family GTPases. p21 activated kinase 4 (PAK4), an effector of the Rho family protein Cdc42, is activated downstream of HGF. We report here the novel finding that in prostate cancer cells PAK4 binds to and phosphorylates LIM kinase 1 (LIMK1) in an HGF-dependent manner. We show for the first time that variations in the level of PAK4 expression change the level of cofilin phosphorylation in cells, a change we correlate with LIMK1 activity, cell morphology and migratory behaviour. We identify for the first time a direct and localised interaction between PAK4 and LIMK1 within cells using FRET: FLIM. Moreover we show here that HGF mediates this interaction which is concentrated in small foci at the cell periphery. PAK4 and LIMK1 act synergistically to increase cell migration speed, whilst a reduction in PAK4 expression decreases cell speed. It is well established that unphosphorylated (active) cofilin is a required to drive cell migration. Our results support a model whereby HGF-stimulated cell migration also requires a cofilin phosphorylation step that is mediated by PAK4.

Primary cultures of renal proximal tubule cells derived from individuals with primary hyperoxaluria.

The primary hyperoxalurias, PH1 and PH2, are inherited disorders caused by deficiencies of alanine:glyoxylate aminotransferase and glyoxylate reductase, respectively. Mutations in either of these enzymes leads to endogenous oxalate overproduction primarily in the liver, but most pathological effects are exhibited in the kidney ultimately leading to end-stage renal failure and systemic oxalosis. To provide a non-invasive means of accessing kidney cells from individuals with primary hyperoxaluria, we have derived primary cultures of renal proximal tubule cells from the urine of these patients. The cells stain positively for the epithelial markers pan-cytokeratin and zonula occludens 1 and the proximal tubule marker gamma-glutamyl transpeptidase. Mutation analysis confirmed that the cultured cells had the same genotype as the leucocytes of the patients and also expressed glyoxylate reductase at the mRNA level, illustrating their potential value as a source of renal material from these individuals.

Elevation of XPA protein level in testis tumor cells without increasing resistance to cisplatin or UV radiation.

Most testicular germ cell tumors are curable using cisplatin-based chemotherapy, and cell lines from these tumors are unusually sensitive to cisplatin and other DNA-damaging agents. It has been suggested that this might be caused by a lower-than normal nucleotide excision repair (NER) activity. Previous studies found that cell lines from testicular germ cell tumors have on average about one-third the level of the NER protein XPA in comparison to cell lines from other tumors. We asked whether over-expression of XPA protein would alleviate the cellular sensitivity and increase the DNA repair capacity of a testis tumor cell line. Increasing XPA levels in 833K cells by 10-fold did not increase resistance to UV irradiation. XPA was localized to the cell nucleus in all cell lines, before and after exposure to UV-radiation. 833K cells were proficient in removing UV radiation-induced photoproducts from the genome and increased XPA did not enhance the rate of removal. Further, over-expressing functional XPA protein did not correlate with increased resistance of 833K testis tumor cells to cisplatin. Thus, although the amount of XPA in this testis tumor cell line is lower than normal, it is sufficient for NER in vivo. The relative sensitivity of testis tumor cells to cisplatin, UV radiation, and other DNA damaging agents is likely related not to NER capacity, but to other factors such as the integrity of the p53 pathway in these cells.

Allelic imbalance at 13q14.2 approximately q14.3 in localized prostate cancer is associated with early biochemical relapse.

Allelic imbalance (AI), particularly at chromosomes 8p, 10q, and 13q, is the most frequently observed genetic change in sporadic prostate cancer. AI at these sites may inactivate tumor suppressor genes that regulate normal cell growth. To establish the relationship between AI and progression, we analyzed loci on 8p, 10q, and 13q14 in archival prostate tumors matched for Gleason grade, pre-operative prostate-specific antigen levels, and pathologic stage, and they were paired on the basis of relapse status after 3 years. AI was identified in 66% of patients without relapse and in 73% with relapse. There was no statistically significant difference for AI at 8p21.3 and 10q23.2 between the two groups of patients, but significant differences between relapsers and nonrelapsers in the frequency of AI at D13S165 at 13q14.2 (P=0.006) and D13S273 at 13q14.3 (P=0.03). There was also a significantly higher incidence of AI at both loci in the relapsers compared to the nonrelapsers (P=0.03). In three relapsers, AI occurred at all three loci between 13q14.2 and 13q14.3, with no nonrelapsers demonstrating AI at all three loci. These findings show that AI at 13q14.2 approximately q14.3 is an important event in the progression of localized prostate cancer, and suggest a possible role for microRNAs.

Clinical applications of expression profiling and proteomics in prostate cancer.

Expression profiling and proteomics have the potential to transform the management of prostate cancer, identifying new markers for screening, diagnosis, prognosis, monitoring and targets for therapy. Expression profiling has revealed that the majority of prostate cancers contain fusion genes resulting in the upregulation of ETS family transcription factors. New diagnostic markers to replace PSA are being actively sought using a variety of proteomic platforms. Nevertheless, no single molecular marker has yet been discovered that is any more reliable for predicting outcome than histopathological grading. In the future, small custom-built chips will be used to detect a small panel of RNA or protein markers to answer specific questions concerning patient management for each type of cancer.

Combined affinity labelling and mass spectrometry analysis of differential cell surface protein expression in normal and prostate cancer cells.

Differences in the expression of cell surface proteins between a normal prostate epithelial (1542-NP2TX) and a prostate cancer cell line (1542-CP3TX) derived from the same patient were investigated. A combination of affinity chromatographic purification of biotin-tagged surface proteins with mass spectrometry analysis identified 26 integral membrane proteins and 14 peripheral surface proteins. The findings confirm earlier reports of altered expression in prostate cancer for several cell surface proteins, including ALCAM/CD166, the Ephrin type A receptor, EGFR and the prostaglandin F2 receptor regulatory protein. In addition, several novel findings of differential expression were made, including the voltage-dependent anion selective channel proteins Porin 1 and 2, ecto-5'-nucleotidase (CD73) and Scavenger receptor B1. Cell surface protein expression changed both qualitatively and quantitatively when the cells were grown in the presence of either or both interferon INFalpha and INFgamma. Costimulation with type I and II interferons had additive or synergistic effects on the membrane density of several, mainly peripherally attached surface proteins. Concerted upregulation of surface exposed antigens may be of benefit in immuno-adjuvant-based treatment of interferon-responsive prostate cancer. In conclusion, this study demonstrates that differences in the expression of membrane proteins between normal and prostate cancer cells are reproducibly detectable following vectorial labelling with biotin, and that detailed analysis of extracellular-induced surface changes can be achieved by combining surface-specific labelling with high-resolution two-dimensional gel electrophoresis and mass spectrometry.

XPA versus ERCC1 as chemosensitising agents to cisplatin and mitomycin C in prostate cancer cells: role of ERCC1 in homologous recombination repair.

Nucleotide excision repair is the principal mechanism for the removal of bulky DNA adducts caused by a range of chemotherapeutic drugs, and contributes to cisplatin resistance. In this study, we used synthetic siRNAs targeted to XPA and ERCC1 and compared their effectiveness in sensitising mismatch repair deficient prostate cancer cell lines to cisplatin and mitomycin C. Downregulation of ERCC1 sensitised DU145 and PC3 cells to cisplatin and mitomycin C. In contrast, XPA downregulation did not sensitise either cell line to mitomycin C, and only sensitised DU145 cells to cisplatin. The effects of ERCC1 downregulation may be due to its role in homologous recombination repair. Excision repair of cisplatin adducts in PC3 cells was attenuated to a similar extent by XPA and ERCC1 downregulation. Downregulation of XPA but not ERCC1 caused an increase in the number of cisplatin-induced RAD51 foci in PC3 cells, suggesting that HRR is able to substitute for NER in these cells. We observed co-localisation of ERCC1 and RAD51 in cisplatin treated PC3 cells by immunofluorescence and co-immunoprecipitation, which may represent recruitment of ERCC1/XPF to sites of recombination repair. These results indicate that ERCC1 is a broader therapeutic target than XPA with which to sensitise cancer cells to chemotherapy because of its additional role in recombination repair.

Quantitative Expression and Co-Localization of Wnt Signalling Related Proteins in Feline Squamous Cell Carcinoma.

Feline oral squamous cell carcinoma (FOSCC) is an aggressive neoplasm in cats. Little is known about the possible molecular mechanisms that may be involved in the initiation, maintenance and progression of FOSCC. Wnt signalling is critical in development and disease, including many mammalian cancers. In this study, we have investigated the expression of Wnt signalling related proteins using quantitative immunohistochemical techniques on tissue arrays. We constructed tissue arrays with 58 individual replicate tissue samples. We tested for the expression of four key Wnt/ß-catenin transcription targets, namely Cyclin D1 (CCND1 or CD1), FRA1, c-Myc and MMP7. All antibodies showed cross reactivity in feline tissue except MMP7. Quantitative immunohistochemical analysis of single proteins (expressed as area fraction / amount of tissue for normal vs tumor, mean ± SE) showed that the expression of CD1 (3.9 ± 0.5 vs 12.2 ± 0.9), FRA1 (5.5 ± 0.6 vs 16.8 ± 1.1) and c-Myc (5.4 ± 0.5 vs 12.5 ± 0.9) was increased in FOSCC tissue by 2.3 to 3 fold compared to normal controls (p<0.0001). By using a multilabel, quantitative fluorophore technique we further investigated if the co-localization of these proteins (all transcription factors) with each other and in the nucleus (stained with 4',6-diamidino-2-phenylindole, DAPI) was altered in FOSCC compared to normal tissue. The global intersection coefficients, a measure of the proximity of two fluorophore labeled entities, showed that there was a significant change (p < 0.01) in the co-localization for all permutations (e.g. CD1/FRA1 etc), except for the nuclear localization of CD1. Our results show that putative targets of Wnt signalling transcription are up-regulated in FOSCC with alterations in the co-localization of these proteins and could serve as a useful marker for the disease.