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Microarray Based Gene Expression Analysis in Cancer Research
KTH, School of Biotechnology (BIO).
2006 (English)Doctoral thesis, comprehensive summary (Other scientific)
Abstract [en]

Biotechnological inventions during the 20th century have resulted in a wide range of approaches for explorations in the functional genomics field. Microarray technology is one of the recent advances which have provided us with snapshots of which genes are expressed in cells of various tissues and diseases. Methods to obtain reliable microarray data are continuously being developed and improved to meet the demands of biological researchers.

In this thesis microarrays have been used to investigate gene expression patterns in cancer research. Four studies in three different areas were carried out covering adrenocortical tumors, p53 target genes and a comparison of RNA amplification methods.

Adrenocortical tumours are among the most common tumours with an incidence of 7-9%. Malignancy of these tumors is rare. Distinction between malignant and benign tumours is often difficult to establish which makes an improvement of diagnostic approaches important. To elucidate biological processes in adrenocortical tumour development and to examine if there is a molecular signature associated with malignancy, microarray analysis was performed on 29 adrenocortical tumors and four normal specimens. It was possible to classify malignant and benign samples based on the entire expression profile. A number of potential biomarkers was identified which will be further evaluated.

P53 is a gene which is mutated in 50% of all cancers. Functional p53 is a transcription factor which is activated upon cellular stress and DNA damage. Target genes are mainly involved in cell cycle arrest and apoptosis. In solid tumors cells are stressed by hypoxia. To examine which target genes p53 activate under hypoxic conditions a microarray study of the cell lines HCT116p53+/+ and HCT116p53-/- was performed. A set of novel potential p53 target genes was identified while many known target genes were found to be not transcriptionally activated during hypoxia. Follow up which was focused on how p53 affected hypoxia induced apoptosis showed that the death receptor Fas was critical.

When small amounts of tissue are available, amplification of the transcript population is necessary for microarray analysis. A new strategy for amplification based on PCR was evaluated and compared to a commercial in vitro transcription protocol. Both protocols produced reliable results. Advantages with the PCR based method are a lower cost and a high flexibility due to compatibility with both sense and antisense strand microarrays.

Keywords: adrenocortical tumour, apoptosis, cancer, classification, gene expression, microarray, p53, RNA amplification

Place, publisher, year, edition, pages
Stockholm: KTH , 2006. , 76 p.
Keyword [ar]
adrenocortical tumour, apoptosis, cancer, classification, gene expression, microarray, p53, RNA amplification
National Category
Cell and Molecular Biology
Identifiers
URN: urn:nbn:se:kth:diva-4244ISBN: 91-7178-542-6 (print)ISBN: 978-91-7178-542-8 (print)OAI: oai:DiVA.org:kth-4244DiVA: diva2:11426
Public defence
2006-12-22, FD5, AlbaNova Universitetscentrum, Roslagstullsbacken 21, Stockholm, 10:00
Opponent
Supervisors
Note
QC 20100907Available from: 2006-12-15 Created: 2006-12-15 Last updated: 2010-09-07Bibliographically approved
List of papers
1. Comparative analysis of a 3' end tag PCR and a linear RNA amplification approach for microarray analysis
Open this publication in new window or tab >>Comparative analysis of a 3' end tag PCR and a linear RNA amplification approach for microarray analysis
2007 (English)In: Journal of Biotechnology, ISSN 0168-1656, E-ISSN 1873-4863, Vol. 127, no 4, 638-646 p.Article in journal (Refereed) Published
Abstract [en]

 Background: Various types of amplification techniques have been developed in order to enable microarray gene expression analysis when the amount of starting material is limited. The two main strategies are linear amplification, using in vitro transcription, and exponential amplification, based on PCR. We have evaluated the performance of a linear and an in-house developed exponential amplification protocol that relies on 3' end tag sequences. We used 100 ng total RNA as starting material for amplification and compared the results with data from hybridizations with unamplified mRNA and total RNA.

Results: Preservation of expression ratios after amplification was examined comparing 1092 ratios obtained with amplification protocols to those obtained with standard labelling of mRNA. The Pearson correlations were 0.61 and 0.84, respectively, for the two linear amplification replicates and 0.76 and 0.80 for the two exponential amplification replicates. The correlations between repeated amplifications was 0.82 with the exponential method and 0.63 with the linear, indicating a better reproducibility with the PCR-based approach.

Conclusion: Both amplification methods generated results in agreement with unamplified material. In this study, the PCR-based method was more reproducible than in vitro transcription amplification. Advantages with the in-house developed method are the lower cost since it is non-commercial and that the PCR generated product offers compatibility with both sense and antisense arrays.

Keyword
microarray, linear amplification, exponential amplification, RNA amplification, in vitro transcription, PCR
National Category
Biological Sciences
Identifiers
urn:nbn:se:kth:diva-6652 (URN)10.1016/j.jbiotec.2006.08.016 (DOI)000243704600008 ()2-s2.0-33845759906 (Scopus ID)
Note
QC 20100903Available from: 2006-12-15 Created: 2006-12-15 Last updated: 2017-12-14Bibliographically approved
2. Hypoxia induces p53-dependent transactivation and Fas/CD95-dependent apoptosis
Open this publication in new window or tab >>Hypoxia induces p53-dependent transactivation and Fas/CD95-dependent apoptosis
Show others...
2007 (English)In: Cell Death and Differentiation, ISSN 1350-9047, E-ISSN 1476-5403, Vol. 14, no 3, 411-421 p.Article in journal (Refereed) Published
Abstract [en]

p53 triggers apoptosis in response to cellular stress. We analyzed p53-dependent gene and protein expression in response to hypoxia using wild-type p53-carrying or p53 null HCT116 colon carcinoma cells. Hypoxia induced p53 protein levels and p53-dependent apoptosis in these cells. cDNA microarray analysis revealed that only a limited number of genes were regulated by p53 upon hypoxia. Most classical p53 target genes were not upregulated. However, we found that Fas/CD95 was significantly induced in response to hypoxia in a p53-dependent manner, along with several novel p53 target genes including ANXA1, DDIT3/ GADD153 (CHOP), SEL1L and SMURF1. Disruption of Fas/CD95 signalling using anti-Fas-blocking antibody or a caspase 8 inhibitor abrogated p53-induced apoptosis in response to hypoxia. We conclude that hypoxia triggers a p53-dependent gene expression pattern distinct from that induced by other stress agents and that Fas/CD95 is a critical regulator of p53-dependent apoptosis upon hypoxia.

Keyword
p53, hypoxia, apoptosis, microarray analysis, p53 target genes
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-6653 (URN)10.1038/sj.cdd.4402022 (DOI)000244275400004 ()2-s2.0-33847077136 (Scopus ID)
Note

QC 20100907

Available from: 2006-12-15 Created: 2006-12-15 Last updated: 2017-12-14Bibliographically approved
3. Expression profiling of adrenocortical neoplasms suggests a molecular signature of malignancy.
Open this publication in new window or tab >>Expression profiling of adrenocortical neoplasms suggests a molecular signature of malignancy.
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2005 (English)In: Surgery, ISSN 0039-6060, E-ISSN 1532-7361, Surgery, Vol. 138, no 6, 1087-1094 p.Article in journal (Refereed) Published
Abstract [en]

Background. Distinguishing between adrenocortical adenomas and carcinomas is often difficult. Our aim was to investigate the differences in transcriptional profiles between benign and malignant adrenocortical neoplasms using complementary DNA microarray techniques.

Methods. We studied 7 patients with adrenocortical carcinomas and 13 with adenomas. Histopathology was reviewed in all patients, clinical follow-up was at least 1 year. Hybridizations were Performed in duplicate against RNA reference. Expression levels were analyzed in the R environment for statistical computing with the use of aroma, limma, statistics, and class packages.

Results. Transcriptional profiles were homogeneous among adenomas, while carcinomas were much more heterogeneous. Hierarchical clustering and self-organizing maps could separate clearly carcinomas from adenomas. Among genes that were most significantly upregulated in carcinomas were 2 ubiquilin-related genes (USP4 and UFD1L) and several insulinlike growth factor-related genes (IGF2, IGF2R, IGFBP3 and IGFBP6). Among genes that were most significantly downregulated in carcinomas were a cylokine gene (CXCL10), several genes related to cell metabolism (RARRES2, ALDH1A1, CYBRD1 and GSTA4), and the cadherin 2 gene (CDH2).

Conclusions. Through the use of cDNA arrays, adrenocortical adenomas and carcinomas appear to be clearly distinguishable on the basis of their specific molecular signature. The biologic importance of the up- and downregulated genes is yet to be determined.

Keyword
factor-binding protein-3, cancer-risk, tumors, gene, carcinoma, identification, frequent, markers, locus
National Category
Industrial Biotechnology Cell and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-6654 (URN)10.1016/j.surg.2005.09.031 (DOI)000234319000032 ()2-s2.0-29144437760 (Scopus ID)
Note
QC 20100907Available from: 2006-12-15 Created: 2006-12-15 Last updated: 2017-12-14Bibliographically approved
4. Transcriptional profiling enables molecular classification of adrenocortical tumours
Open this publication in new window or tab >>Transcriptional profiling enables molecular classification of adrenocortical tumours
Show others...
2009 (English)In: European Journal of Endocrinology, ISSN 0804-4643, E-ISSN 1479-683X, Vol. 161, no 1, 141-152 p.Article in journal (Refereed) Published
Abstract [en]

Objective: Tumours in the adrenocortex are common human tumours. Malignancy is however, rare, the yearly incidence being 0.5-2 per million inhabitants, but associated with a very aggressive behaviour. Adrenocortical tumours are often associated with altered hormone production with a variety of clinical symptoms. The aggressiveness of carcinomas together with the high frequency of adenomas calls for a deeper understanding of the underlying biological mechanisms and an improvement of the diagnostic possibilities. Methods: Microarray gene expression analysis was performed in tumours of adrenocortex with emphasis on malignancy as well as hormonal activity. The sample set consisted of 17 adenomas, 11 carcinomas and 4 histological normal adrenocortexes. RNA from these was hybridised according to a reference design on microarrays harbouring 29 760 human cDNA clones. Confirmation was performed with quantitative real time-PCR and western blot analysis. Results: Unsupervised clustering to reveal relationships between samples based on the entire gene expression profile resulted in two subclusters; carcinomas and non-cancer specimens. A large number of genes were accordingly found to be differentially expressed comparing carcinomas to adenomas. Among these were IGF2, FGFR1 and FGFR4 in growth factor signalling the most predominant and also the USP4, UBE2C and UFD1L in the ubiquitin-proteasome pathway. Moreover, two subgroups of carcinomas were identified with different survival outcome, suggesting that survival prediction can be made on the basis of gene expression profiles. Regarding adenomas with aldosterone overproduction, OSBP and VEGFB were among the most up-regulated genes compared with the other samples. Conclusions: Adrenocortical carcinomas are associated with a distinct molecular signature apparent in their gene expression profiles. Differentially expressed genes were identified associated with malignancy, survival as well as hormonal activity providing a resource of candidate genes for an exploration of possible drug targets and diagnostic and prognostic markers.

Keyword
ubiquitin-specific protease, gene-expression, carcinoma, cancer, malignancy, unp, enzyme, tumorigenesis, protooncogene, microarrays
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:kth:diva-6655 (URN)10.1530/EJE-09-0068 (DOI)000272934700018 ()19411298 (PubMedID)2-s2.0-67650736272 (Scopus ID)
Funder
Swedish Research Council
Note
QC 20100907 Uppdaterad från manuskript till artikel (20100907).Available from: 2006-12-15 Created: 2006-12-15 Last updated: 2017-12-14Bibliographically approved

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