Heart failure and apoptosis: Electrophoretic methods support data from micro- and macro-arrays.

1. Heart failure and apoptosis:Electrophoretic methods support data from micro- and macro-arrays. 생명과학부 이 수 민

2. The multiple cause and multiple consequences of mammalian heart failure make it an attractive proposition for analysis using gene array technology. • However, gene array also hold potential problem • Also, recently doubts were raised about the qualitative reliability of gene array • Electrophoretic methods are slow, cumbersome and complex but they can provide confirmation

3. In this overview, they compare gene array data with data from protein assays such as zymograms, western blots, two-dimensional electrophoresis, and immunohistochemistry. • Similar principles will apply to other tissues and cells.

4. contents 1.What are gene arrays? 1.1 Noncompetive hybridization gene arrays 1.2 competive gene arrays 1.3 Macroarray 1.4 Rehability of gene array technology 1.5 Visualization and quantification of the hybridized arrays 1.6 How many chip experiments are needed? 1.7 How do data from different array compare? 1.8 Why “invest” in gene arrays?

5. 2 The proteomics world 2.1 2-DE analysis of failing mammalian hearts 2.2 Comparison of data from 2-DE and gene arrays 2.3 Functional change in proteins 2.4 Western blots 2.5 Comparison of apoptosis data from gene arrays and Western blots 2.6 Identifying the cells responsible for gene an protein changes 3 TUNEL assays 4 Cell capture assays 5 The future

6. 1.What are gene arrays? • Gene arrays are microdots of nucleic acids forming a deposited area 50-100µm in diameter • cDNAs are usually placed on coated glass slides such that potentially the gamut of human genes might be arrayed on single slide. • The cDNA can be either full length gene or short oligonucleotide

7. 1.1 Noncompetitve hybridization gene arrays • The Affymetrix Hugene FL gene chip uses short oligomers • Labelled cDNA probe is applied to the surface of the chip and hybridized with complementary sequence. • Mismatches between the target cDNAs and the probe array cDNAs produce weaker signals • Given that the sequence and the location of the probe on the array are known, the identity of the target cDNA can be determined.

9. 1.2 Competitive gene arrays • New England Nuclear employs long cDNAs • They routinely contain 2400 human genes • The NEN 2400 system requires the isolation of total RNA from two separate sample populations. • These are converted to cDNAs and labelled • For example, a control cDNA can be labelled with Cy3-dUTP, while an experimental sample is separately labelled with Cy5-dUTP.

10. Both control and experimental labelled cDNAs are are mixed together and hybridized on the chip overnight, and the resulting florescent chip scanned and the data analyzed.

11. 1.3 Macroarrays • Macroarrays are another class of gene arrays where cDNAs are dotted onto nylon membranes. • The detection was based the radioactivity of 32P or 33P which is more sensitive than flourescence detection. • Another feature of the nylon arrays is that they can be re-used 3-4 times.

13. 1.4 Reliability of gene array technology • As powerful as the gene array technology may be, the reliability of gene arrays is still a major concern. • Both competitive and non competitive gene arrays technologies may contain inaccuracies • This is caused by wrong sequence information in the databases as well as errors generated during manufacturing.

14. However, we should not be too discouraged by this since the technology is still in its “infancy” • We can expect major technology improvements. • Also we can reduce inaccuracies on the arrays by “ checking the sequence of the spot concerned and verifying the result using alternative methods monitoring gene expression”

15. 1.5 Visualization and quantification of the hybridized arrays • The cyanine dyes are effective, but where expression level are low, other methods can be employed. • Also, other dyes are now being developed. • Figure 1 is pie-chart display of cy3 and cy5 location of gene on the array. • Figure 2 illustrates a macroarray for human apoptosis gene.

18. 1.6 How many chip experiments are needed? • Expression differences of less than 2.0-fold 2.5-fold may not be significant. • Finally, but no means the least important, is question of how to extract “significant” information from amongst the vast array of data. • “Data mining” is rapidly evolving but it seems fair to say that current software will only improve. • Public domain software is available • GeneSpring is one of the leading software to analyze and visualize genomic expression data

19. 1.7 How do data from different arrays compare? • In competitive arrays we found only 10 genes that up-regulated whereas 60 were down-regulated. • Alpha-B-crystallin(2.5-fold down in competitive array. 2.3-fold down in noncompetitive array) • Succinate dehydrogenase flavoprotein subunit

20. 1.8 Why “invest” in gene arrays? • Commercial gene arrays, when they first appeared on the scientific market, were at once a bargain and a great expense. • A single gene array can provide information that will inexorably lead to new insights which can then be pursued by more conventional assay • What other genes change in concerted way? • How can these genes be identified?

21. 2 The proteomics world • The so-called post-genomics era in which we now live is likely to remain relevant as the era of proteomics • The interaction between gene arrays and proteomics will also intensify.

22. 2.1 2-DE analysis of failing mammalian hearts • It may even be possible to resolve each of five to ten thousand proteins expressed by a tissue using a large-format 2-DE gel. • However, the similarities between gene arrays and 2-DE are modest. • There are a few variation. • Staining is not perfectly uniform nor do all proteins stain with equal intensity. • As a result, the same sample should be run on multiple gels and under identical conditions.

23. They identified 23 proteins either from a dog cardiac protein database. • Fig. 3. On the basis of these data, we concluded that heart failure was associated with shifts in three cellular system: (1) mitochondrial proteins which were down-regulated. (2) glycolytic enzymes were up-regulated (3) changes in the cytoskeletal proteins.

25. 2.2 comparison of data from 2-DE and gene arrays • The proteins listed by NEN provided a match of six of the 23 proteins identified by 2-DE. • When a protein appeared to be either up-regulated or down-regulated on 2-DE gels, it was usually similarly regulated on the gene arrays. • However, the magnitudes of the changes did not agree closely.

27. 2.3 Functional changes in proteomics • Cell extracts containing DNase I can be run on standard SDS-PAGE mini gels in the presence DNA, the SDS is then removed, the DNase I in gel re-natures and resumes its activity. • It is clear that DNase I is up-regulated in the failing heart sample. • We were unable to confirm changes in Dnase I in either of the microarrays or in 2-DE gels. • However, we know that Dnase I is associated with apoptosis

29. Accordingly we searched both the 2-DE gel database and the gene array data for evidence of apoptosis. • Only two of the 62 proteins were likely to be involved in apoptosis. • The arrays revealed that two mRNAs were up-regulated.

31. 2.4 Western blots • The western blot is a standard method of identifying the presence or absence of proteins in a tissue sample. • Quantification of western blots can be difficult • Some of these difficulties can be controlled, for example by comparing standard proteins, or by comparing a spot density with the integrated total spot densities in a given gel lane

32. 2.5 Comparison of apoptosis data from gene arrays Western blots • Within this context of uncertainly, Table 2 compares data from Western blots using two animal models with gene array data from human left ventricles. • Several proteins are known components of apoptotic pathways. • The up-regulated genes are activators of apoptosis. • One gene (Bcl-2) is an inhibitor of apoptosis that it was down-regulated. • These findings are consistent with a large volume of literature implicating apoptosis in heart failure.

33. 3 TUNEL assays • Apoptosis can be demonstrated in cardiomyocytes using TUNEL assay • Activation of DNases produces regularly spaced cleavage of nuclear DNA and the subsequent exposed 3’ends of the DNA which can then be labelled with a flurorescent probe, 12-dUTP-Cy3.

35. 4. Cell capture assays • New instrutments are currently available that enable the examination of a tissue section and the subsequent capture of a specific single cell or small group. • A laser beam to melt the cells of interest onto plastic film. • The total mRNA content of these cells can then be amplified (RT-PCR) and the gene expression interrogated. • We have not yet accessed this technology. • But clearly it represents yet another advance that can be correlated with gene arrays.

36. 5 The future • A few years ago, molecular biology was the most popular research technique. • Protein chemists were largely frowned upon as unfashionable • Today, the pendulum is swing back to protein. • In this brief report, we have gathered together a variety of different kinds of techniques that focus apoptosis in failure heart • We conclude that gene microarrays and macroarrays can broadly evaluate the expression of these genes. • There is a strong need to greatly expand the numbers of cDNAs avaliable on the microarrays. • It should then be possible to correlate gene clanges with protein alterations.

37. Where the changes in genes involves a disease process, we must then speculate whether particular gene is causative in development of a disease. • Electrophoretic methods will be needed more than ever.