Cluster analyses of the TCGA and a TMA dataset using the coexpression of HSP27 and CRYAB improves alignment with clinical-pathological parameters of breast cancer and suggests different epichaperome influences for each sHSP.

Our cluster analysis of the Cancer Genome Atlas for co-expression of HSP27 and CRYAB in breast cancer patients identified three patient groups based on their expression level combination (high HSP27 + low CRYAB; low HSP27 + high CRYAB; similar HSP27 + CRYAB). Our analyses also suggest that there is a statistically significant inverse relationship between HSP27 and CRYAB and known clinicopathological markers in breast cancer. Screening an unbiased 248 breast cancer patient tissue microarray (TMA) for the protein expression of HSP27 and phosphorylated HSP27 (HSP27-82pS) with CRYAB also identified three patient groups based on HSP27 and CRYAB expression levels. TMA24 also had recorded clinical-pathological parameters, such as ER and PR receptor status, patient survival, and TP53 mutation status. High HSP27 protein levels were significant with ER and PR expression. HSP27-82pS associated with the best patient survival (Log Rank test). High CRYAB expression in combination with wild-type TP53 was significant for patient survival, but a different patient outcome was observed when mutant TP53 was combined with high CRYAB expression. Our data suggest that HSP27 and CRYAB have different epichaperome influences in breast cancer, but more importantly evidence the value of a cluster analysis that considers their coexpression. Our approach can deliver convergence for archival datasets as well as those from recent treatment and patient cohorts and can align HSP27 and CRYAB expression to important clinical-pathological features of breast cancer.

Microarray analysis of bleomycin-exposed lymphoblastoid cells for identifying cancer susceptibility genes.

The uncovering of genes involved in susceptibility to the sporadic cancer types is a great challenge. It is well established that the way in which an individual deals with DNA damage is related to the chance to develop cancer. Mutagen sensitivity is a phenotype that reflects an individual's susceptibility to the major sporadic cancer types, including colon, lung, and head and neck cancer. A standard test for mutagen sensitivity is measuring the number of chromatid breaks in lymphocytes after exposure to bleomycin. The aim of the present study was to search for the pathways involved in mutagen sensitivity. Lymphoblastoid cell lines of seven individuals with low mutagen sensitivity were compared with seven individuals with a high score. RNA was isolated from cells exposed to bleomycin (4 hours) and from unexposed cells. Microarray analysis (19K) was used to compare gene expression of insensitive and sensitive cells. The profile of most altered genes after bleomycin exposure, analyzed in all 14 cell lines, included relatively many genes involved in biological processes, such as cell growth and/or maintenance, proliferation, and regulation of cell cycle, as well as some genes involved in DNA repair. When comparing the insensitive and sensitive individuals, other differentially expressed genes were found that are involved in signal transduction and cell growth and/or maintenance (e.g., BUB1 and DUSP4). This difference in expression profiles between mutagen-sensitive and mutagen-insensitive individuals justifies further studies aimed at elucidating the genes responsible for the development of sporadic cancers.

BAC to the future! or oligonucleotides: a perspective for micro array comparative genomic hybridization (array CGH).

The array CGH technique (Array Comparative Genome Hybridization) has been developed to detect chromosomal copy number changes on a genome-wide and/or high-resolution scale. It is used in human genetics and oncology, with great promise for clinical application. Until recently primarily PCR amplified bacterial artificial chromosomes (BACs) or cDNAs have been spotted as elements on the array. The large-scale DNA isolations or PCR amplifications of the large-insert clones necessary for manufacturing the arrays are elaborate and time-consuming. Lack of a high-resolution highly sensitive (commercial) alternative has undoubtedly hindered the implementation of array CGH in research and diagnostics. Recently, synthetic oligonucleotides as arrayed elements have been introduced as an alternative substrate for array CGH, both by academic institutions as well as by commercial providers. Oligonucleotide libraries or ready-made arrays can be bought off-the-shelf saving considerable time and efforts. For RNA expression profiling, we have seen a gradual transition from in-house printed cDNA-based expression arrays to oligonucleotide arrays and we expect a similar transition for array CGH. This review compares the different platforms and will attempt to shine a light on the 'BAC to the future' of the array CGH technique.

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.

In vivo induction of resistance to gemcitabine results in increased expression of ribonucleotide reductase subunit M1 as the major determinant.

Gemcitabine is a deoxycytidine (dCyd) analogue with activity against several solid cancers. Gemcitabine is activated by dCyd kinase (dCK) and interferes, as its triphosphate dFdCTP, with tumor growth through incorporation into DNA. Alternatively, the metabolite gemcitabine diphosphate (dFdCDP) can interfere with DNA synthesis and thus tumor growth through inhibition of ribonucleotide reductase. Gemcitabine can be inactivated by the enzyme dCyd deaminase (dCDA). In most in vitro models, resistance to gemcitabine was associated with a decreased dCK activity. In all these models, resistance was established using continuous exposure to gemcitabine with increasing concentrations; however, these in vitro models have limited clinical relevance. To develop in vivo resistance to gemcitabine, we treated mice bearing a moderately sensitive tumor Colon 26-A (T/C = 0.25) with a clinically relevant schedule (120 mg/kg every 3 days). By repeated transplant of the most resistant tumor and continuation of gemcitabine treatment for >1 year, the completely resistant tumor Colon 26-G (T/C = 0.96) was created. Initial studies focused on resistance mechanisms known from in vitro studies. In Colon 26-G, dCK activity was 1.7-fold decreased; dCDA and DNA polymerase were not changed; and Colon 26-G accumulated 1.5-fold less dFdCTP, 6 hours after a gemcitabine injection, than the parental tumor. Based on in vitro studies, these relative minor changes were considered insufficient to explain the completely resistant phenotype. Therefore, an expression microarray was done with Colon 26-A versus Colon 26-G. Using independently grown nonresistant and resistant tumors, a striking increase in expression of the RRM1 subunit gene was found in Colon 26-G. The expression of RRM1 mRNA was 25-fold increased in the resistant tumor, as measured by real-time PCR, which was confirmed by Western blotting. In contrast, RRM2 mRNA was 2-fold decreased. However, ribonucleotide reductase enzyme activity was only moderately increased in Colon 26-G. In conclusion, this is the first model with in vivo induced resistance to gemcitabine. In contrast to most in vitro studies, dCK activity was not the most important determinant of gemcitabine resistance. Expression microarray identified RRM1 as the gene with the highest increase in expression in the Colon 26-G, which might clarify its complete gemcitabine-resistant phenotype. This study is the first in vivo evidence for a key role for RRM1 in acquired gemcitabine resistance.

Desmin aggregate formation by R120G alphaB-crystallin is caused by altered filament interactions and is dependent upon network status in cells.

The R120G mutation in alphaB-crystallin causes desmin-related myopathy. There have been a number of mechanisms proposed to explain the disease process, from altered protein processing to loss of chaperone function. Here, we show that the mutation alters the in vitro binding characteristics of alphaB-crystallin for desmin filaments. The apparent dissociation constant of R120G alphaB-crystallin was decreased while the binding capacity was increased significantly and as a result, desmin filaments aggregated. These data suggest that the characteristic desmin aggregates seen as part of the disease histopathology can be caused by a direct, but altered interaction of R120G alphaB-crystallin with desmin filaments. Transfection studies show that desmin networks in different cell backgrounds are not equally affected. Desmin networks are most vulnerable when they are being made de novo and not when they are already established. Our data also clearly demonstrate the beneficial role of wild-type alphaB-crystallin in the formation of desmin filament networks. Collectively, our data suggest that R120G alphaB-crystallin directly promotes desmin filament aggregation, although this gain of a function can be repressed by some cell situations. Such circumstances in muscle could explain the late onset characteristic of the myopathies caused by mutations in alphaB-crystallin.

Nuclear speckle localisation of the small heat shock protein alpha B-crystallin and its inhibition by the R120G cardiomyopathy-linked mutation.

In this study, the small heat shock protein (sHSP) chaperones, alpha B-crystallin and HSP27, are identified as nuclear speckle components in unstressed cells in tissue culture. This new finding suggests a constitutive function for these sHSP chaperones in the nucleus and suggests a new perspective on the cardiomyopathy-causing mutation for alpha B-crystallin that could involve transcriptional splicing effects. Both alpha B-crystallin and HSP27 were immunolocalised to nuclear speckles (interchromatin granule clusters). While alpha B-crystallin was preferentially localised to speckles as shown by colocalisation with non-snRNP, SC35, as well as the snRNP components Sm and U1A, HSP27 was also seen associated with the nucleolar compartment, indicating a subtle difference between these closely related sHSPs. Actinomycin D treatment caused the relocalisation of alpha B-crystallin along with Sm and SC35 to a smaller number of more distinct spots, suggesting a link between speckle localisation and the transcriptional status of the cells. We then examined several transformed, immortalised, and primary cells expressing endogenous alpha B-crystallin as well as some cells with ectopic alpha B-crystallin expression. All consistently showed alpha B-crystallin in nuclear speckles. The nuclear localisation of the sHSPs was also confirmed biochemically and 2D gel electrophoresis revealed that there was only one major nuclear alpha B-crystallin isoform. This suggested that phosphorylation was not required for nuclear localisation of alpha B-crystallin. This was confirmed by the transient transfection of HeLa cells with a phosphorylation-defective alpha B-crystallin. In contrast, the transfection of R120G alpha B-crystallin, the mutation that causes cardiomyopathy, inhibited the nuclear speckle localisation of alpha B-crystallin. These data suggest that the cardiomyopathy-causing mutation for alpha B-crystallin has nuclear as well as cytoplasmic consequences, suggesting an explanation for the difference in severity of the desmin and alpha B-crystallin transgenic models of their respective cardiomyopathies.

Differential effect of simvastatin on activation of Rac(1) vs. activation of the heat shock protein 27-mediated pathway upon oxidative stress, in human smooth muscle cells.

In the present study, we have analyzed the response of human smooth muscle cell (SMC)s to oxidative stress, in terms of recruitment of key elements of the stress-activated protein kinase (SAPK) pathway, such as Rac(1), p38, and the small heat shock protein (HSP)27. The level of expression of three small HSPs, alphaB-crystallin, HSP20, HSP27, as well as the phosphorylation levels of HSP27 and p38, were higher in cultured, asynchronously growing SMCs originating from left interior mammary artery (LIMA) than those originating from aorta, saphenous vein, and umbilical vein, validating the choice of SMCs from LIMA as a model system in our study. In synchronized, quiescent SMCs from LIMA, oxidative stress (H(2)O(2) stimulation)-induced membrane translocation of Rac(1), p38 phosphorylation, membrane translocation, and phosphorylation of HSP27. In these cells, simvastatin (S), an HMG-CoA reductase inhibitor, blocked, in a mevalonate-dependent way, oxidative stress-induced membrane translocation of Rac(1). However, S pretreatment prior to oxidative stress increased the levels of p38 phosphorylation, HSP27 membrane translocation/phosphorylation, actin polymerization, and apoptosis in these cells, in a mevalonate-dependent way. These results establish that S pretreatment has a stimulatory effect on the stress-activated p38/HSP27 pathway, despite its blocking effect on Rac(1) activation.

Translocation of small heat shock proteins to the actin cytoskeleton upon proteasomal inhibition.

The role of small heat shock proteins (sHsps) as molecular chaperones is still poorly understood. We therefore investigated the effect of proteasomal inhibition on sHsps in the rat cardiac myoblast cell line H9c2. Proteasomes are responsible for controlled degradation of intracellular proteins. Inhibition of their activities leads to accumulation of unfolded proteins, which can form insoluble "aggresomes" together with proteasomes and heat shock proteins Hsp70 and Hsp90. We here report that upon proteasome inhibition, alpha B-crystallin and Hsp25 translocate from the detergent-soluble cytosolic fraction to the detergent-insoluble nuclear/cytoskeletal fraction. Although phosphorylation of both alpha B-crystallin and Hsp25 is induced, this does not seem to be essential for the translocation. Immunocytochemistry revealed that alpha B-crystallin and Hsp25, which show a diffuse cytoplasmic staining in unstressed H9c2 cells, colocalize with F-actin upon proteasomal inhibition. After transfection in H9c2 cells, other sHsps (alpha A-crystallin, Hsp20, HspB2 and HspB3) showed similar translocation to the actin cytoskeleton. The redistribution of sHsps upon proteasomal inhibition may reflect a mechanism by which cells are protected from damaged intracellular proteins by sequestering them on the cytoskeleton.