Profiling of apoptosis genes allows for clinical stratification of primary nodal diffuse large B-cell lymphomas.

Intrinsic resistance of lymphoma cells to apoptosis is a probable mechanism causing chemotherapy resistance and eventual fatal outcome in patients with diffuse large B cell lymphomas (DLBCL). We investigated whether microarray expression profiling of apoptosis related genes predicts clinical outcome in 46 patients with primary nodal DLBCL. Unsupervised cluster analysis using genes involved in apoptosis (n = 246) resulted in three separate DLBCL groups partly overlapping with germinal centre B-lymphocytes versus activated B-cells like phenotype. One group with poor clinical outcome was characterised by high expression levels of pro-and anti-apoptotic genes involved in the intrinsic apoptosis pathway. A second group, also with poor clinical outcome, was characterised by high levels of apoptosis inducing cytotoxic effector genes, possibly reflecting a cellular cytotoxic immune response. The third group showing a favourable outcome was characterised by low expression levels of genes characteristic for both other groups. Our results suggest that chemotherapy refractory DLBCL are characterised either by an intense cellular cytotoxic immune response or by constitutive activation of the intrinsic mediated apoptosis pathway with concomitant downstream inhibition of this apoptosis pathway. Consequently, strategies neutralising the function of apoptosis-inhibiting proteins might be effective as alternative treatment modality in part of chemotherapy refractory DLBCL.

Increased gene copy numbers at chromosome 20q are frequent in both squamous cell carcinomas and adenocarcinomas of the cervix.

Genome-wide microarray-based comparative genomic hybridization (array CGH) was used to identify common chromosomal alterations involved in cervical carcinogenesis as a first step towards the discovery of novel biomarkers. The genomic profiles of nine squamous cell carcinomas (SCCs) and seven adenocarcinomas (AdCAs), as well as four human papillomavirus (HPV)-immortalized keratinocyte cell lines, were assessed. On a genome-wide scale, SCCs showed significantly more gains than AdCAs. More specifically, there was a striking and highly significant difference between the two histological types for gain at 3q12.1-28, which was predominantly observed in SCC. Other frequent alterations included gains of 1q21.1-31.1 and 20q11.21-13.33, and losses of 11q22.3-25 and 13q14.3-21.33. Subsequent FISH analysis for hTR, located at 3q26, confirmed the presence of 3q gain in SCCs and HPV-immortalized cell lines. Fine mapping of chromosome 20q using multiplex ligation-dependent probe amplification (MLPA) showed copy number increases for a number of genes located at 20q11-q12, including DNMT3B and TOP1. For DNMT3B, this correlated with elevated mRNA expression in 79% of cases. In conclusion, the assessment of frequent genomic alterations resulted in the identification of potential novel biomarkers, which may ultimately enable a better risk stratification of high-risk (hr)-HPV-positive women.

Phosphorylation of alphaB-crystallin and HSP27 is induced by similar stressors in HeLa cells.

Three members of the small heat shock protein family, alphaA-, alphaB-crystallin, and HSP27, confer thermoresistance upon their overexpression in mammalian cells. Phosphorylation, in conjunction with the molecular chaperone-like activity of these small HSPs, is believed to be important for this in situ functional property. We here report the influence of heat shock and other kinds of stress on the phosphorylation of alphaA-, alphaB-crystallin, and HSP27 in stably transfected HeLa cells. It is observed that alphaB-crystallin becomes phosphorylated upon exposure to the same inducers as is HSP27, although to a lesser extent. In contrast, phosphorylation of alphaA-crystallin is very low upon heat stress and even absent when other stressors are used. This indicates that phosphorylation is not in all instances essential for the stress protective functioning of the various small HSPs.

alpha B-crystallin and hsp25 in neonatal cardiac cells--differences in cellular localization under stress conditions.

Two members of the small heat shock protein family, alpha B-crystallin and hsp25, occur at high levels in the mammalian heart. To try and understand any differences in functioning, we compared their properties in cultured rat neonatal cardiac myocytes. Both proteins are stress-inducible, but the level of hsp25 is only slightly increased in cultured cardiac myocytes subjected to hyperthermic stress, while alpha B-crystallin levels even remain unchanged. Phosphorylation of alpha B-crystallin and to a lesser extent also of hsp25 is induced after the heat shock. Directly after heat stress, alpha B-crystallin and hsp25 are partly found in detergent-insoluble fractions, representing cytoskeletal/nuclear structures. Additionally, we show by confocal laser scanning microscopy that alpha B-crystallin and hsp25 become associated with sarcomeric structures directly after the heat shock, indicating a cytoskeletal protective function. Four to six hours after the heat shock, both proteins reoccupy their original positions in the cytoplasm again. In contrast to alpha B-crystallin, hsp25 not only translocates to the cytoskeleton but also migrates to positions inside the nucleus. Despite the fact that both proteins are normally part of the same complex, their behavior in neonatal cardiac myocytes appears to be very different. The sarcomeric association of alpha B-crystallin occurs under milder conditions and persists for a longer period of time in comparison with hsp25. Our findings suggest that alpha B-crystallin and hsp25 are both involved in protection of the cytoskeleton during stress situations in the heart, although in different manners. In addition, hsp25 also plays a role inside the nucleus.

alpha-Crystallin: molecular chaperone and heat shock protein.

The relationship of alpha-crystallin with the family of small heat shock proteins has led to the discovery that the basic subunit alpha B-crystallin can, like other heat shock proteins, protect cells against heat stress. Here we show that the acidic subunit alpha A-crystallin, which in contrast to alpha B-crystallin is expressed mainly in the eye lens, shares this property. Furthermore we have investigated the in vitro molecular chaperone-like behavior of the natural mutant alpha A ins-crystallin that has a large insert peptide and occurs in rodents. We have found the chaperone-like activity of the mutant to be diminished compared to that of the wild type alpha A-crystallin.

Molecular analysis of glyceraldehyde-3-phosphate dehydrogenase in Trypanoplasma borelli: an evolutionary scenario of subcellular compartmentation in kinetoplastida.

In Trypanoplasma borelli, a representative of the Bodonina within the Kinetoplastida, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) activity was detected in both the cytosol and glycosomes. This situation is similar to that previously found in Trypanosomatidae, belonging to a different Kinetoplastida suborder. In Trypanosomatidae different isoenzymes, only distantly related, are responsible for the activity in the two cell compartments. In contrast, immunoblot analysis indicated that the GAPDH activity in cytosol and glycosomes of T. borelli should be attributed to identical or at least very similar proteins related to the glycosomal GAPDH of Trypanosomatidae. Moreover, only genes related to the glycosomal GAPDH genes of Trypanosomatidae could be detected. All attempts to identify a gene related to the one coding for the trypanosomatid cytosolic GAPDH remained unsuccessful. Two tandemly arranged genes were found which are 95% identical. The two encoded polypeptides differ in 17 residues. Their sequences are 72-77% identical to the glycosomal GAPDH of the other Kinetoplastida and share with them some characteristic features: an excess of positively charged residues, specific insertions, and a small carboxy-terminal extension containing the sequence -AKL. This tripeptide conforms to the consensus signal for targeting of proteins to glycosomes. One of the two gene copies has undergone some mutations at positions coding for highly conserved residues of the active site and the NAD(+)-binding domain of GAPDH. Modeling of the protein's three-dimensional structure suggested that several of the substitutions compensate each other, retaining the functional coenzyme-binding capacity, although this binding may be less tight. The presented analysis of GAPDH in T. borelli gives further support to the assertion that one isoenzyme, the cytosolic one, was acquired by horizontal gene transfer during the evolution of the Kinetoplastida, in the lineage leading to the suborder Trypanosomatina (Trypanosoma, Leishmania), after the divergence from the Bodonina (Trypanoplasma). Furthermore, the data clearly suggest that the original GAPDH of the Kinetoplastida has been compartmentalized during evolution.

Alpha A-crystallin confers cellular thermoresistance.

The bovine eye lens protein alpha A-crystallin has been overexpressed both by stable transfection of HeLa cells and by transient transfection of NIH 3T3 cells. In both experimental systems alpha A-crystallin overexpression results in an increased cellular thermoresistance as judged by different clonal survival assays. In contrast, similar overexpression of another stable lens protein, beta B2-crystallin, does not confer thermoresistance. These results indicate that the structural relationship of alpha A-crystallin to the small heat shock proteins HSP25/27 and to alpha B-crystallin is sufficient for the shared thermoprotective function of all of these molecules and strongly suggests that the chaperone-like properties that they have in common are responsible for the conferred cellular thermoresistance.

The amine-donor substrate specificity of tissue-type transglutaminase. Influence of amino acid residues flanking the amine-donor lysine residue.

The amine-donor substrate specificity of tissue-type transglutaminase has been studied in a series of recombinant alpha A-crystallin mutants. These mutant proteins have been provided with a potential substrate lysine residue, flanked by different amino acid residues, in the C-terminal extended arm of alpha A-crystallin. A biotinylated amine-acceptor hexapeptide was used as a probe for labelling the amine-donor sites. Wild-type bovine alpha A-crystallin does not function as an amine-donor substrate for tissue-type transglutaminase. Yet, upon introduction of a lysine residue at the C-terminal or penultimate position, all mutant alpha A-crystallins act as amine-donor substrates, although to different extents. This shows that accessibility is the primary requirement for a lysine residue to function as an amine-donor substrate for transglutaminase and that the enzyme has a broad tolerance towards the neighbouring residues. However, the nature of the flanking amino acid residues does clearly affect the reactivity of the substrate lysine residue. Notably, we found that a proline or glycine residue in front of the substrate lysine has a strong adverse effect on the substrate reactivity as compared to a preceding leucine, serine, alanine or arginine residue.

Site-specific racemization in aging alpha A-crystallin.

Of all aspartyl residues in bovine alpha A-crystallin, only Asp-151 exhibits pronounced racemization. Asp-151 is also one of the sites where peptide bond cleavage occurs in in vivo aging alpha A-crystallin. This aspartyl residue is followed by an alanyl residue and resides in a flexible carboxyl terminal extension of alpha-crystallin. Both in vivo and in vitro racemization studies indicate that the pronounced and site-specific racemization of Asp-151 proceeds via formation of a succinimide intermediate. The in vivo racemization of aspartyl residues in alpha A-crystallin is discussed with regard to the proposed tertiary structure of alpha-crystallin.