Biomedical subjects

Carlos Caldas

Publications and source records attributed to Carlos Caldas.

At least 19 recordsLinked to original sources

PubMed2006

ZNF198, a zinc finger protein rearranged in myeloproliferative disease, localizes to the PML nuclear bodies and interacts with SUMO-1 and PML.

The ZNF198/FGFR1 fusion gene in atypical myeloproliferative disease produces a constitutively active cytoplasmic tyrosine kinase, unlike ZNF198 which is normally a nuclear protein. We have now shown that the ZNF198/FGFR1 fusion kinase interacts with the endogenous ZNF198 protein suggesting that the function of ZNF198 may be compromised in cells expressing it. Little is currently known about the endogenous function of ZNF198 and to investigate this further we performed a yeast two-hybrid analysis and identified SUMO-1 as a binding partner of ZNF198. These observations were confirmed using co-immunoprecipitation which demonstrated that ZNF198 is covalently modified by SUMO-1. Since many of the SUMO-1-modified proteins are targeted to the PML nuclear bodies we used confocal microscopy to show that SUMO-1, PML and ZNF198 colocalize to punctate structures, shown by immunocytochemistry to be PML bodies. Using co-immunoprecipitation we now show that PML and sumoylated ZNF198 can be found in a protein complex in the cell. Mutation of the SUMO-1 binding site in wild-type ZNF198 resulted in loss of distinct PML bodies, reduced PML levels and a more dispersed nuclear localization of the PML protein. In cells expressing ZNF198/FGFR1, which also lack the SUMO-1 binding site, SUMO-1 is preferentially localized in the cytoplasm, which is associated with loss of distinct PML bodies. Recently, arsenic trioxide (ATO) was proposed as an alternative therapy for APL that was resistant to traditional therapy. Treatment of cells expressing ZNF198/FGFR1 with ATO demonstrated reduced autophosphorylation of the ZNF198/FGFR1 protein and induced apoptosis, which is not seen in cells expressing wild-type ZNF198. Overall our results suggest that the sumoylation of ZNF198 is important for PML body formation and that the abrogation of sumoylation of ZNF198 in ZNF198/FGFR1 expressing cells may be an important mechanism in cellular transformation.

Amino Acid Substitution↗

PubMed2006

Metabolic consequences of p300 gene deletion in human colon cancer cells.

Metabolite profiling using (1)H nuclear magnetic resonance (NMR) spectroscopy was used to investigate the metabolic changes associated with deletion of the gene for the transcriptional coactivator p300 in the human colon carcinoma cell line HCT116. Multivariate statistical methods were used to distinguish between metabolite patterns that were dependent on cell growth conditions and those that were specifically associated with loss of p300 function. In the absence of serum, wild-type cells showed slower growth, which was accompanied by a marked decrease in phosphocholine concentration, which was not observed in otherwise isogenic cell lines lacking p300. In the presence of serum, several metabolites were identified as being significantly different between the two cell types, including glutamate and glutamine, a nicotinamide-related compound and glycerophosphocholine (GPC). However, in the absence of serum, these metabolites, with the exception of GPC, were not significantly different, leading us to conclude that most of these changes were context dependent. Transcript profiling, using DNA microarrays, showed changes in the levels of transcripts for several enzymes involved in choline metabolism, which might explain the change in GPC concentration. Localized in vivo (1)H NMR measurements on the tumors formed following s.c. implantation of these cells into mice showed an increase in the intensity of the peak from choline-containing compounds in the p300(-) tumors. These data show that NMR-based metabolite profiling has sufficient sensitivity to identify the metabolic consequences of p300 gene deletion in tumor cells in vitro and in vivo.

Animals↗

PubMed2006

PACK: Profile Analysis using Clustering and Kurtosis to find molecular classifiers in cancer.

MOTIVATION: Elucidating the molecular taxonomy of cancers and finding biological and clinical markers from microarray experiments is problematic due to the large number of variables being measured. Feature selection methods that can identify relevant classifiers or that can remove likely false positives prior to supervised analysis are therefore desirable. RESULTS: We present a novel feature selection procedure based on a mixture model and a non-gaussianity measure of a gene's expression profile. The method can be used to find genes that define either small outlier subgroups or major subdivisions, depending on the sign of kurtosis. The method can also be used as a filtering step, prior to supervised analysis, in order to reduce the false discovery rate. We validate our methodology using six independent datasets by rediscovering major classifiers in ER negative and ER positive breast cancer and in prostate cancer. Furthermore, our method finds two novel subtypes within the basal subgroup of ER negative breast tumours, associated with apoptotic and immune response functions respectively, and with statistically different clinical outcome. AVAILABILITY: An R-function pack that implements the methods used here has been added to vabayelMix, available from (www.cran.r-project.org). CONTACT: aet21@cam.ac.uk SUPPLEMENTARY INFORMATION: Supplementary information is available at Bioinformatics online.

Algorithms↗

PubMed2006

Differential expression of selected histone modifier genes in human solid cancers.

BACKGROUND: Post-translational modification of histones resulting in chromatin remodelling plays a key role in the regulation of gene expression. Here we report characteristic patterns of expression of 12 members of 3 classes of chromatin modifier genes in 6 different cancer types: histone acetyltransferases (HATs)- EP300, CREBBP, and PCAF; histone deacetylases (HDACs)- HDAC1, HDAC2, HDAC4, HDAC5, HDAC7A, and SIRT1; and histone methyltransferases (HMTs)- SUV39H1and SUV39H2. Expression of each gene in 225 samples (135 primary tumours, 47 cancer cell lines, and 43 normal tissues) was analysedby QRT-PCR, normalized with 8 housekeeping genes, and given as a ratio by comparison with a universal reference RNA. RESULTS: This involved a total of 13,000 PCR assays allowing for rigorous analysis by fitting a linear regression model to the data. Mutation analysis of HDAC1, HDAC2, SUV39H1, and SUV39H2 revealed only two out of 181 cancer samples (both cell lines) with significant coding-sequence alterations. Supervised analysis and Independent Component Analysis showed that expression of many of these genes was able to discriminate tumour samples from their normal counterparts. Clustering based on the normalized expression ratios of the 12 genes also showed that most samples were grouped according to tissue type. Using a linear discriminant classifier and internal cross-validation revealed that with as few as 5 of the 12 genes, SIRT1, CREBBP, HDAC7A, HDAC5 and PCAF, most samples were correctly assigned. CONCLUSION: The expression patterns of HATs, HDACs, and HMTs suggest these genes are important in neoplastic transformation and have characteristic patterns of expression depending on tissue of origin, with implications for potential clinical application.

Breast Neoplasms↗

PubMed2006

Bcl-2 is a prognostic marker in breast cancer independently of the Nottingham Prognostic Index.

PURPOSE: Prognostication of breast cancer using clinicopathologic variables, although useful, remains imperfect. Many reports suggest that gene expression profiling can refine the current approach. Alternatively, it has been shown that panels of proteins assessed by immunohistochemistry might also be useful in this regard. We evaluate the prognostic potential of a panel of markers by immunohistochemistry in a large case series to establish if either a single marker or a panel could improve the prognostic power of the Nottingham Prognostic Index (NPI). We validated the results in an independent series. EXPERIMENTAL DESIGN AND RESULTS: The expression of 13 biomarkers was evaluated in 930 breast cancers on a tissue microarray. Eight markers [estrogen receptor (ER), progesterone receptor (PR), Bcl-2, cyclin E, p53, MIB-1, cytokeratin 5/6, and HER2] showed a significant association with survival at 10 years on univariate analysis. On multivariate analysis that included these eight markers and the NPI, only the NPI [hazard ratio (HR), 1.35; 95% confidence interval (95% CI), 1.16-1.56; P = 0.0005], ER (HR, 0.59; 95% CI, 0.39-0.88; P = 0.011), and Bcl-2 (HR, 0.68; 95% CI, 0.46-0.99; P = 0.055) were significant. In a subsequent multivariate analysis that included the NPI, ER, and Bcl-2, only Bcl-2 (HR, 0.62; 95% CI, 0.44-0.87; P = 0.006) remained independent of NPI (HR, 1.50; 95% CI, 1.16-1.56; P = 0.004). In addition, Bcl-2, used as a single marker, was more powerful than the use of a panel of markers. Based on these results, an independent series was used to validate the prognostic significance of Bcl-2. ER and PR were also evaluated in this validation series. Bcl-2 (HR, 0.83; 95% CI, 0.71-0.96; P = 0.018) retained prognostic significance independent of the NPI (HR, 2.04; 95% CI, 1.67-2.51; P < 0.001) with an effect that was maximal in the first 5 years. CONCLUSION: Bcl-2 is an independent predictor of breast cancer outcome and seems to be useful as a prognostic adjunct to the NPI, particularly in the first 5 years after diagnosis.

Adult↗

PubMed2006

Telomerase- and alternative telomere lengthening-independent telomere stabilization in a metastasis-derived human non-small cell lung cancer cell line: effect of ectopic hTERT.

In the majority of human malignancies, maintenance of telomeres is achieved by reactivation of telomerase, whereas a smaller fraction uses an alternative telomere lengthening (ALT) mechanism. Here, we used 16 non-small cell lung cancer (NSCLC) cell lines to investigate telomere stabilization mechanisms and their effect on tumor aggressiveness. Three of 16 NSCLC cell lines (VL-9, SK-LU-1, and VL-7) lacked telomerase activity, correlating with significantly reduced tumorigenicity in vitro and in vivo. Of the three telomerase-negative cell lines, only SK-LU-1 displayed characteristics of an ALT mechanism (i.e., highly heterogeneous telomeres and ALT-associated promyelocytic leukemia bodies). VL-9 cells gained telomerase during in vitro propagation, indicating incomplete immortalization in vivo. In contrast, NSCLC metastasis-derived VL-7 cells remained telomerase and ALT negative up to high passage numbers and following transplantation in severe combined immunodeficient mice. Telomeres of VL-7 cells were homogeneously short, and chromosomal instability (CIN) was comparable with most telomerase-positive cell lines. This indicates the presence of an efficient telomere stabilization mechanism different from telomerase and ALT in VL-7 cells. To test the effect of ectopic telomerase reverse transcriptase (hTERT) in these unique ALT- and telomerase-negative tumor backgrounds, hTERT was transfected into VL-7 cells. The activation of telomerase led to an excessively rapid gain of telomeric sequences resulting in very long ( approximately 14 kb), uniform telomeres. Additionally, hTERT expression induced a more aggressive growth behavior in vitro and in vivo without altering the level of CIN. These data provide further evidence for a direct oncogenic activity of hTERT not based on the inhibition of CIN.

Animals↗

PubMed2006

High-resolution magic angle spinning 1H NMR spectroscopy and reverse transcription-PCR analysis of apoptosis in a rat glioma.

The functional genomic approaches of transcriptomics, proteomics and metabolomics aim to measure the mRNA, protein or metabolite complement of a cell, tissue or organism. In this study we have investigated the compatibility of transcriptional analysis, using Reverse Transcription (RT)-PCR, and metabolite analysis, by high-resolution magic angle spinning (HRMAS) 1H NMR spectroscopy, in BT4C rat glioma following the induction of programmed cell death. The metabolite and transcriptional changes that accompanied apoptosis were examined at 0, 4 and 8 days of ganciclovir/thymidine kinase gene therapy. Despite the high spinning speeds employed during HRMAS 1H NMR spectroscopy of one-half of the tumor samples, RT-PCR analysis of the pro-apoptotic transcripts Bcl-2, BAK-1, caspase-9 and FAS was possible, producing similar results to those detected in the unspun half of the tumors. Furthermore, the expression of FAS was inversely correlated with some of the key metabolic changes across the time period examined including the increases CH=CH and CH=CHCH2 lipid resonances which accompany apoptosis. This study demonstrates how combined transcriptomic and metabolomic studies of tumors can be used to understand the molecular events that accompany well documented metabolic perturbations during cell death processes.

Animals↗

PubMed2006

Pharmacogenomic identification of novel determinants of response to chemotherapy in colon cancer.

DNA microarray analysis was used to analyze the transcriptional profile of HCT116 colorectal cancer cells that were treated with 5-fluorouracil (5-FU) or oxaliplatin and selected for resistance to these agents. Bioinformatic analyses identified sets of genes that were constitutively dysregulated in drug-resistant cells and transiently altered following acute exposure of parental cells to drug. We propose that these genes may represent molecular signatures of sensitivity to 5-FU and oxaliplatin. Using real-time reverse transcription-PCR (RT-PCR), the robustness of our microarray data was shown with a strong overall concordance of expression trends for > or =82% (oxaliplatin) and > or =85% (5-FU) of a representative subset of genes. Furthermore, strong correlations between the microarray and real-time RT-PCR measurements of average fold changes in gene expression were observed for both the 5-FU (R(2) > or = 0.73) and oxaliplatin gene sets (R(2) > or = 0.63). Functional analysis of three genes identified in the microarray study [prostate-derived factor (PDF), calretinin, and spermidine/spermine N(1)-acetyl transferase (SSAT)] revealed their importance as novel regulators of cytotoxic drug response. These data show the power of this novel microarray-based approach to identify genes which may be important markers of response to treatment and/or targets for therapeutic intervention.

Antineoplastic Agents↗

PubMed2006

Genomic imbalances and patterns of karyotypic variability in mantle-cell lymphoma cell lines.

Mantle-cell lymphoma (MCL) is genetically characterized by 11q13 chromosomal translocations involving the CCND1 gene. We have characterized five MCL cell lines, JVM-2, GRANTA-519, REC-1, JEKO-1, and NCEB-1, combining metaphase and array comparative genomic hybridization, multicolor-FISH, and molecular analysis. Our results revealed common gained regions at 2p14, 9q31.2-qter, 11q13.1-q21, 13q14-q21.2, 13q34-qter and 18q21.1-q22.1, and losses at 1p21.2-p31.1, 2p11.2, 8p21.2-pter, 9p21.3-pter, 11q23.3-qter, 17p11.2-pter, and 17q21.2-q22.2. All cell lines except JVM-2, displayed moderate or high numerical chromosome instability. In addition, an ongoing level of chromosome rearrangements was observed in REC-1. Surprisingly, NCEB-1 carried several stable mouse chromosomes and showed expression of both human and murine bcl-2 protein. Our findings indicate that these cell lines represent three patterns of chromosome evolution in MCL and may be useful to understand the pathogenesis of this neoplasm.

Animals↗

PubMed2006

Familial gastric cancer - clinical management.

The clinical management of familial gastric cancer is the same as that for sporadic gastric cancer at the current time. As the causative mutations for these cases are identified this should lead to the development of specific treatments which target the molecular abnormality. The only germline mutations identified so far occur within the E-cadherin gene (CDHI) and they account for approximately 30% of familial gastric cancer cases. When index patients fulfilling the clinical criteria for hereditary diffuse gastric cancer syndrome have a CDHI mutation identified then genetic testing of asymptomatic relatives should be considered. The clinical sequelae of testing positive for such a mutation are profound and therefore it is essential that counselling is given prior to genetic testing. The management options are surveillance endoscopy and prophylactic gastrectomy. In this chapter the practicalities of genetic testing are discussed as well as the pros and cons of the two management options. It is essential that experience of these rare families is pooled so that surveillance and treatment can be optimised in the future.

Endoscopy, Gastrointestinal↗

PubMed2006

Familial gastric cancer - aetiology and pathogenesis.

Gastric cancer is the second most common cause of cancer death worldwide. It is estimated that 5-10% of gastric cancer cases have a familial association; however, knowledge concerning the genetic predisposition to familial gastric cancer is currently limited. In this chapter we discuss what is known about the aetiology and pathogenesis of both the diffuse and intestinal forms of familial gastric cancer. We focus particularly on hereditary diffuse gastric cancer because the discovery of germ-line E-cadherin mutations in a number of affected families has opened the prospect of identifying gene carriers, with implications for clinical management. The interplay of other conventional risk factors, such as Helicobacter pylori infection, with genetic factors is also discussed. It is hoped that understanding the genetic basis for familial gastric cancer will facilitate the development of clinically useful screening and preventative procedures.

Genes, Neoplasm↗

PubMed2005

Human and mouse oligonucleotide-based array CGH.

Array-based comparative genomic hybridization is a high resolution method for measuring chromosomal copy number changes. Here we present a validated protocol using in-house spotted oligonucleotide libraries for array comparative genomic hybridization (CGH). This oligo array CGH platform yields reproducible results and is capable of detecting single copy gains, multi-copy amplifications as well as homozygous and heterozygous deletions as small as 100 kb with high resolution. A human oligonucleotide library was printed on amine binding slides. Arrays were hybridized using a hybstation and analysed using BlueFuse feature extraction software, with >95% of spots passing quality control. The protocol allows as little as 300 ng of input DNA and a 90% reduction of Cot-1 DNA without compromising quality. High quality results have also been obtained with DNA from archival tissue. Finally, in addition to human oligo arrays, we have applied the protocol successfully to mouse oligo arrays. We believe that this oligo-based platform using 'off-the-shelf' oligo libraries provides an easy accessible alternative to BAC arrays for CGH, which is cost-effective, available at high resolution and easily implemented for any sequenced organism without compromising the quality of the results.

Animals↗

PubMed2005

Chromatin modifier enzymes, the histone code and cancer.

In all organisms, cell proliferation is orchestrated by coordinated patterns of gene expression. Transcription results from the activity of the RNA polymerase machinery and depends on the ability of transcription activators and repressors to access chromatin at specific promoters. During the last decades, increasing evidence supports aberrant transcription regulation as contributing to the development of human cancers. In fact, transcription regulatory proteins are often identified in oncogenic chromosomal rearrangements and are overexpressed in a variety of malignancies. Most transcription regulators are large proteins, containing multiple structural and functional domains some with enzymatic activity. These activities modify the structure of the chromatin, occluding certain DNA regions and exposing others for interaction with the transcription machinery. Thus, chromatin modifiers represent an additional level of transcription regulation. In this review we focus on several families of transcription activators and repressors that catalyse histone post-translational modifications (acetylation, methylation, phosphorylation, ubiquitination and SUMOylation); and how these enzymatic activities might alter the correct cell proliferation program, leading to cancer.

Acetylation↗

PubMed2005

Chromosome abnormalities in 10 lung cancer cell lines of the NCI-H series analyzed with spectral karyotyping.

The karyotypes of 10 lung cancer cell lines of the NCI-H series were analyzed with spectral karyotyping (SKY): 7 non-small lung cancer (NSCLC) lines and 3 small cell lung cancer (SCLC) lines. Modal chromosome number ranged from 42 (NCI-H2171) to 72 (NCI-H2126). All lines showed at least six structural abnormalities, and most had amplifications visible as double minutes or homogeneously staining regions (HSRs). Four reciprocal translocations were found: t(1;17)(p10;p10) in NCI-H82, t(3;6)(q24;q21) and t(12;17)(p10;p10) in NCI-H2009, and a complex t(2;6) in NCI-H1437. NCI-H1770 had a striking HSR containing many copies of the NMYC region. Karyotypes showed a wide range of relationship between numerical and structural change. Two of the lines showed little numerical change but many structural rearrangements (NCI-H209 with mode 46, but 12 rearrangements, and NCI-H2009 with mode 48 but 27 rearrangements). A second group had karyotypes that appeared to have evolved by unbalanced translocation leading to proportionate loss of chromosomes, with or without endoreduplication. In other lines, notably NCI-H2122, the structurally abnormal chromosomes appeared to have been added to a near-diploid karyotype. The karyotypes contribute to a full genomic characterization of these lines, almost all of which have matching normal lymphoblastoid cell lines.

Adenocarcinoma↗

PubMed2005

Molecular classification and molecular forecasting of breast cancer: ready for clinical application?

Profiling breast cancer with expression arrays has become common, and it has been suggested that the results from early studies will lead to understanding of the molecular differences between clinical cases and allow individualization of care. We critically review two main applications of expression profiling; studies unraveling novel breast cancer classifications and those that aim to identify novel markers for prediction of clinical outcome. Breast cancer may now be subclassified into luminal, basal, and HER2 subtypes with distinct differences in prognosis and response to therapy. However, profiling studies to identify predictive markers have suffered from methodologic problems that prevent general application of their results. Future work will need to reanalyze existing microarray data sets to identify more representative sets of candidate genes for use as prognostic signatures and will need to take into account the new knowledge of molecular subtypes of breast cancer when assessing predictive effects.

Biomarkers, Tumor↗

PubMed2005

A 1 Mb minimal amplicon at 8p11-12 in breast cancer identifies new candidate oncogenes.

Amplification of 8p11-12 is a well-known alteration in human breast cancers but the driving oncogene has not been identified. We have developed a high-resolution comparative genomic hybridization array covering 8p11-12 and analysed 33 primary breast tumors, 20 primary ovarian tumors and 27 breast cancer cell lines. Expression analysis of the genes in the region was carried out by using real-time quantitative PCR and/or oligo-microarray profiling. In all, 24% (8/33) of the breast tumors, 5% (1/20) of the ovary tumors and 15% (4/27) of the cell lines showed 8p11-12 amplification. We identified a 1 Mb segment of common amplification that excludes previously proposed candidate genes. Some of the amplified genes did not show overexpression, whereas for others, overexpression was not specifically attributable to amplification. The genes FLJ14299, C8orf2, BRF2 and RAB11FIP, map within the 8p11-12 minimal amplicon, two have a putative function consistent with an oncogenic role, these four genes showed a strong correlation between amplification and overexpression and are therefore the best candidate driver oncogenes at 8p12.

Breast Neoplasms↗

PubMed2005

A variational Bayesian mixture modelling framework for cluster analysis of gene-expression data.

MOTIVATION: Accurate subcategorization of tumour types through gene-expression profiling requires analytical techniques that estimate the number of categories or clusters rigorously and reliably. Parametric mixture modelling provides a natural setting to address this problem. RESULTS: We compare a criterion for model selection that is derived from a variational Bayesian framework with a popular alternative based on the Bayesian information criterion. Using simulated data, we show that the variational Bayesian method is more accurate in finding the true number of clusters in situations that are relevant to current and future microarray studies. We also compare the two criteria using freely available tumour microarray datasets and show that the variational Bayesian method is more sensitive to capturing biologically relevant structure.

Algorithms↗

PubMed2005

Loss of acetylation at Lys16 and trimethylation at Lys20 of histone H4 is a common hallmark of human cancer.

CpG island hypermethylation and global genomic hypomethylation are common epigenetic features of cancer cells. Less attention has been focused on histone modifications in cancer cells. We characterized post-translational modifications to histone H4 in a comprehensive panel of normal tissues, cancer cell lines and primary tumors. Using immunodetection, high-performance capillary electrophoresis and mass spectrometry, we found that cancer cells had a loss of monoacetylated and trimethylated forms of histone H4. These changes appeared early and accumulated during the tumorigenic process, as we showed in a mouse model of multistage skin carcinogenesis. The losses occurred predominantly at the acetylated Lys16 and trimethylated Lys20 residues of histone H4 and were associated with the hypomethylation of DNA repetitive sequences, a well-known characteristic of cancer cells. Our data suggest that the global loss of monoacetylation and trimethylation of histone H4 is a common hallmark of human tumor cells.

Acetylation↗