Hypoxia induces the expression of transketolase-like 1 in human colorectal cancer.

BACKGROUND AND AIMS: Transketolase-like (TKTL) 1 is one of the key enzymes for anaerobic sugar degradation even in the presence of oxygen (aerobic glycolysis). Transketolase-dependent reactions supply malignant tumors with ribose and NADPH. Therefore, TKTL1 activity could be crucial for tumor proliferation and survival. The aim of the study was to evaluate the expression of TKTL1 in colorectal cancer (CRC) and its regulation under hypoxic conditions. METHODS: We studied TKTL1 mRNA and protein expression in CRC cell lines and human CRC biopsies by quantitative real-time PCR, Western blotting and immunohistochemistry. Regulation of TKTL1 under oxygen depletion was analyzed by cultivating cells either in a three-dimensional spheroid model or in a hypoxia incubator chamber. RESULTS: TKTL1 mRNA was heterogeneously expressed in monolayers of cells with high levels in HT-29 and SW480. TKTL1 protein was also clearly detectable in HT-29 and SW480. Hypoxia-inducible factor (HIF)-1α protein expression correlated with TKTL1 protein expression in SW480 spheroids over time. On the one hand, induction of hypoxia in T84 spheroids did not induce TKTL1; on the other hand, hypoxia by incubation at 1% O2 in a hypoxia incubator chamber clearly showed an upregulation of TKTL1. In 50% of CRC patients, TKTL1 protein expression was upregulated in tumor compared to non-tumor tissue. The immunohistochemical staining of TKTL1 in CRC patient samples resulted in 14 positive and 30 negative samples. CONCLUSIONS: TKTL1 expression correlated with HIF-1α protein expression and was induced upon hypoxic conditions which could facilitate energy supply to tumors under these circumstances.

Transketolase-like 1 expression correlates with subtypes of ovarian cancer and the presence of distant metastases.

Tumorbiology of ovarian cancer remains unclear. However, it is known that ovarian tumors, especially carcinomas, show elevated expression of glucose membrane transporters for facilitated glucose uptake. It can be assumed that increased glucose uptake leads to higher glucose metabolism. The energy resources of fully malignant transformed carcinomas are mainly supplied by aerobic glycolysis, for which several pathways are known. A key role in aerobic glycolysis is described for the transketolase enzymes. Recently, a novel transketolase-like enzyme called transketolase-like 1 (TKTL1) has been described that links aerobic glycolysis to the synthesis of fatty acids via production of acetyl-CoA. In order to investigate the role of TKTL1 for the progression of ovarian carcinomas, we examined paraffin sections of normal ovarian tissues, ovarian borderline tumors, and mucinous or serous papillary ovarian adenocarcinomas with respect to their expression of TKTL1. We identified a significantly elevated expression of TKTL1 in serous papillary ovarian adenocarcinomas, which correlates with poor prognostic parameters in the examined study group. Therefore, it can be assumed that TKTL1 plays a crucial role in ovarian cancer metabolism and that its expression predicts poor prognosis. Further investigations should be performed in order to evaluate whether this new enzyme is important for ovarian cancer tumorbiology and to analyze the potential role of TKTL1 as new target for specific antitumoral therapy.

Expression of transketolase TKTL1 predicts colon and urothelial cancer patient survival: Warburg effect reinterpreted.

Tumours ferment glucose to lactate even in the presence of oxygen (aerobic glycolysis; Warburg effect). The pentose phosphate pathway (PPP) allows glucose conversion to ribose for nucleic acid synthesis and glucose degradation to lactate. The nonoxidative part of the PPP is controlled by transketolase enzyme reactions. We have detected upregulation of a mutated transketolase transcript (TKTL1) in human malignancies, whereas transketolase (TKT) and transketolase-like-2 (TKTL2) transcripts were not upregulated. Strong TKTL1 protein expression was correlated to invasive colon and urothelial tumours and to poor patients outcome. TKTL1 encodes a transketolase with unusual enzymatic properties, which are likely to be caused by the internal deletion of conserved residues. We propose that TKTL1 upregulation in tumours leads to enhanced, oxygen-independent glucose usage and a lactate-based matrix degradation. As inhibition of transketolase enzyme reactions suppresses tumour growth and metastasis, TKTL1 could be the relevant target for novel anti-transketolase cancer therapies. We suggest an individualised cancer therapy based on the determination of metabolic changes in tumours that might enable the targeted inhibition of invasion and metastasis.

Genetic discordance between primary tumours and metastases of head and neck cancer detected by microsatellite analysis.

Very little is known about possible intra-tumoural genetic heterogeneity between primary tumours and lymph node metastases in head and neck squamous cell carcinoma (HNSCC). To investigate this phenomenon, we analysed 96 micro-dissected tumour samples for allelic imbalance at four of the most frequently altered chromosomal locations in HNSCC (3p14.2; 9p21; 11q23.3; 17p13.1) using microsatellite markers. From 23 patients, matched pairs of primary tumour and lymph node metastasis were analysed. Discordance in the allelic distribution was identified in 8 cases (35%). With one exception, the metastasis contained a more balanced allelic status than the primary tumour. In contrast, in a group of 25 tumours with two anatomically different samples from the primary tumour site, discordance was identified in only 3 tumours (13%). These results are compatible with the dissemination of subclones from the primary tumour site with a more balanced allelotype in the metastasis. In our opinion, several scenarios could explain this phenomenon. From a clinical point of view, genetic discordance between the metastasis and the primary tumour must be taken into consideration when establishing molecular biologic markers for choice of therapy and prognosis in head and neck cancer.

The relationship between allelic imbalance on 17p, p53 mutation and p53 overexpression in head and neck cancer.

The huge majority of head and neck squamous cell carcinoma (HNSCC) show alterations of p53 either on the genetic level or on the protein level. Allelic imbalance (AI)/loss of heterozygosity (LOH) on 17p at the p53 locus is frequent in HNSCC. However, the complex relationship between these phenomena is poorly understood in HNSCC. We investigated one group of 39 HNSCC for: a) allelic imbalance on 17p using 4 microsatellite markers located throughout this chromosomal arm; b) mutations of p53 in exons 5-9; and c) overexpression of p53 using two antibodies located on opposite ends of the protein. AI/LOH was detected in 44% at the locus TP53, rising to 69% when regarding all 4 markers on 17p. Therefore, our data are in line with the assumption of additional tumour suppressor genes on 17p in HNSCC. A nuclear accumulation of p53 (51%) was independent from the antibody and the recognised epitope. At the first glance there was no correlation between overall p53 mutation (36%) and overexpression. However, it appeared that, with very few exceptions, only nonsense mutations did not lead to p53 overexpression, while missense mutations did. As overexpression of p53 was 15% more frequent than p53 mutations and only 35% of the tumours with p53 overexpression carried a p53 mutation, our data support the hypothesis of additional mechanisms of p53 overexpression. AI/LOH at the p53 locus in 83% of all tumours with a p53 mutation is in line with Knudson's theory of inactivation of tumour suppressor genes.

Differential transcriptional regulation of the monocyte-chemoattractant protein-1 (MCP-1) gene in tumorigenic and non-tumorigenic HPV 18 positive cells: the role of the chromatin structure and AP-1 composition.

The expression of the monocyte-chemoattractant-protein-1 (MCP-1) is closely linked with a non-tumorigenic phenotype in somatic cell hybrids made between the human papillomavirus type 18 (HPV 18) positive cervical carcinoma cell line HeLa and normal human fibroblasts. In contrast, MCP-1 transcription is absent in tumorigenic segregants derived from the same hybrids or in parental HeLa cells. Selectivity of MCP-1 transcription, which is regulated at the level of initiation of transcription, is mainly based on differences in the location and extension of DNAse I-hypersensitive regions (DHSR) at both ends of the gene. While TNF-alpha only moderately increases the sensitivity of pre-existing 5'-DHSRs, a 3'-end DHSR became strongly induced exclusively in non-malignant hybrids. DNA sequencing showed that the 3'-DHSR coincides with an additional AP-1 site located approximately 600 bp downstream of the polyadenylation site. Analyses of AP-1 composition revealed that MCP-1 is only expressed in those cells where jun-family members were mainly heterodimerized with the fos-related protein fra-1. In contrast, in tumorigenic cells the 1: 1 ratio between jun and fra-1 is disturbed and the MCP-1 gene is no longer expressed. Hence, alterations in the heterodimerization pattern of AP-1 and its selective accessibility to opened chromatin may represent a novel regulatory pathway in the regulation of chemokines in malignant and non-malignant HPV-positive cells.

Salivary gland carcinosarcoma: immunohistochemical, molecular genetic and electron microscopic findings.

Salivary gland carcinosarcoma, or true malignant mixed tumor, is a very rare and extremely aggressive neoplasm. The clonality and clonal origin of this tumor are discussed controversially. We report a carcinosarcoma of the left parotid gland in a patient who subsequently died of cutaneous, lymphatic and pulmonary metastases. Immunohistochemical staining, electron micrograph analysis, loss of heterozygosity (LOH) analysis and sequence analysis were performed on this tumor with an adenocarcinomatous and a predominant spindle cell-like component. While smooth muscle actin was undetectable by immunohistochemistry, cytoplasmatic myoepithelial structures could be detected by electron microscopy. LOH analysis at 12 genomic locations detected complete deletion of one allele at 17p13.1, 17q21. 3, and 18q21.3 indicating allelic loss in both components of the tumor. Double strand sequencing of the remaining allele of the p53 tumor suppressor gene revealed a wild-type allele. Based on our results, we favor the hypothesis of monoclonal origin of this salivary gland carcinosarcoma with a common stem cell that could be the myoepithelial cell and an inactivated tumor suppressor gene on chromosome 17 other than p53.

Functional characterization of DNase X, a novel endonuclease expressed in muscle cells.

The activation of endonucleases resulting in the degradation of genomic DNA is one of the most characteristic changes in apoptosis. Here, we report the characterization of a novel endonuclease, termed DNase X due to its X-chromosomal localization. The active nuclease is a 35 kDa protein with 39% identity to DNase I. When incubated with isolated nuclei, recombinant DNase X was capable of triggering DNA degradation at internucleosomal sites. Similarly to DNase I, the nuclease activity of DNase X was dependent on Ca(2+) and Mg(2+) and inhibited by Zn(2+) ions or chelators of bivalent cations. Overexpression of DNase X caused internucleosomal DNA degradation and induction of cell death associated with increased caspase activation. Despite the presence of two potential caspase cleavage sites, DNase X was processed neither in vitro nor in vivo by different caspases. Interestingly, after initiation of apoptosis DNase X was translocated from the cytoplasm to the nuclear compartment and aggregated as a detergent-insoluble complex. Abundant expression of DNase X mRNA was detected in heart and skeletal muscle cells, suggesting that DNase X may be involved in apoptotic or other biological events in muscle tissues.

Fhit expression is absent or reduced in a subset of primary head and neck cancer.

BACKGROUND: The inactivation of the FHIT gene at 3p14.2 by various mechanisms might be of importance in head and neck squamous cell carcinoma (HNSCC). Most reports are based on DNA and RNA findings of intragenic deletions and abnormal transcripts. MATERIAL AND METHODS: To study the protein expression of this putative tumour suppressor gene, we analysed 48 HNSCCs by immunohistochemistry using a polyclonal antibody (ZR44). The results were compared with mutation analysis, clinical data and loss of heterozygosity (LOH) data at 3p14.2. RESULTS: Complete absence of Fhit expression was detected in 8 out of 48 of tumours (17%) and 3 tumours (6%) showed heterogenous staining. The overall frequency of LOH for microsatellite D3S1234 was 64% and 5/7 of Fhit negative tumours exhibited LOH. CONCLUSION: Our findings provide further evidence that FHIT is inactivated in a subtype of HNSCC; however, the incidence of lack of Fhit expression compared to the high frequency of LOH on chromosome 3p supports the notion of additional tumour suppressor genes at 3p14.

The genomic structure of the DMBT1 gene: evidence for a region with susceptibility to genomic instability.

Increasing evidence has accumulated for an involvement of the inactivation of tumour suppressor genes at chromosome 10q in the carcinogenesis of brain tumours, melanomas, and carcinomas of the lung, the prostate, the pancreas, and the endometrium. The gene DMBT1 (Deleted in Malignant Brain Tumours 1) is located at chromosome 10q25.3-q26.1, within one of the putative intervals for tumour suppressor genes. DMBT1 is a member of the scavenger-receptor cysteine-rich (SRCR) superfamily and displays homozygous deletions or lack of expression in glioblastoma multiforme, medulloblastoma, and in gastrointestinal and lung cancers. Based on these properties, DMBT1 has been proposed to be a candidate tumour suppressor gene. We have determined the genomic sequence of DMBT1 to allow analyses of mutations. The gene has at least 54 exons that span a genomic region of about 80 kb. We have identified a putative exon with coding potential for a transmembrane domain. Our data further suggest that alternative splicing gives rise to isoforms of DMBT1 with a differential utilization of SRCR domains and SRCR interspersed domains. The major part of the gene harbours locus specific repeats. These repeats may point to the DMBT1 locus as a region susceptible to chromosomal instability.

A complex pattern of evolutionary conservation and alternative polyadenylation within the long 3"-untranslated region of the methyl-CpG-binding protein 2 gene (MeCP2) suggests a regulatory role in gene expression.

A systematic search for expressed sequences in the human Xq28 region resulted in the isolation of 8.5 kb large contigs of human and murine cDNAs with no apparent conserved open reading frames. These cDNAs were found to be derived from the 3"-untranslated region (3"-UTR) of the methyl-CpG-binding protein 2 gene ( MeCP2 ). This long 3"-UTR is part of an alternatively polyadenylated, 10.1 kb MeCP2 transcript which is differentially expressed in human brain and other tissues. RNA in situ hybridization to sections of mouse embryo and adult tissues of an Mecp2 3"-UTR probe showed ubiquitous low level expression in early organogenesis and enhanced expression in the hippocampus during formation of the differentiated brain. Sequence comparison between the human and mouse homologues revealed several blocks of very high conservation separated by less conserved sequences. Additional support for a domain-like conservation pattern of the long 3"-UTR of the MeCP2 gene was obtained by examining conservation in the chimpanzee, orangutan, macaque, hamster, rat and kangaroo. The minimum free energy distribution for the predicted RNA secondary structure was very similar in human and mouse sequences. In particular, the conserved blocks were predicted to be of high minimum free energy, which suggests weak secondary structure with respect to RNA folding. The fact that both the sequence and predicted secondary structure have been highly conserved during evolution suggests that both the primary sequence and the three-dimensional structure of the 3"-UTR may be important for its function in post-transcriptional regulation of MeCP2 expression.

Molecular cloning and characterization of the cDNA encoding the largest subunit of mouse RNA polymerase I.

We describe the cloning and analysis of mRPA1, the cDNA encoding the largest subunit (RPA194) of murine RNA polymerase I. The coding region comprises an open reading frame of 5151 bp that encodes a polypeptide of 1717 amino acids with a calculated molecular mass of 194 kDa. Alignment of the deduced protein sequence reveals homology to the beta' subunit of Escherichia coli RNA polymerase in the conserved regions a-h present in all large subunits of RNA polymerases. However, the overall sequence homology among the conserved regions of RPA1 from different species is significantly lower than that observed in the corresponding beta'-like subunits of class II and III RNA polymerase. We have raised two types of antibodies which are directed against the conserved regions c and f of RPA194. Both antibodies are monospecific for RPA194 and do not cross-react with subunits of RNA polymerase II or III. Moreover, these antibodies immunoprecipitate RNA polymerase I both from murine and human cell extracts and, therefore, represent an invaluable tool for the identification of RNA polymerase I-associated proteins.

Cloning and cytotoxicity of a human pancreatic RNase immunofusion.

BACKGROUND: Immunotoxins based on plant and bacterial proteins are usually very immunogenic. Human ribonucleases could provide an alternative basis for the construction of less immunogenic reagents. Two members of the human RNase family, angiogenin and eosinophil-derived neurotoxin (EDN), have been fused to a single chain antibody against the transferrin receptor, which is known to be internalised by endocytosis. The fusion proteins proved to be very efficient inhibitors of protein synthesis using various cell lines. It is not yet known whether the side effects of angiogenin and EDN will compromise their potential use as immunotoxins. OBJECTIVES: The goal of this work was to construct a human immunotoxin with no harmful side effects. Bovine pancreatic ribonuclease has been shown to be as potent as ricin at abolishing protein synthesis on injection into oocytes. We therefore decided to clone its human analogue, which is fairly ubiquitous and per se non-toxic. An immunofusion of human pancreatic RNase with a single chain antibody against the transferrin receptor was tested for its ability to inhibit protein synthesis in three different human tumor cell lines. STUDY DESIGN: DNA coding for the human pancreatic RNase was cloned partially from a human fetal brain cDNA library and then completed by PCR using a human placental cDNA library as a template. The RNase gene was then fused with a DNA coding for an single chain antibody against the transferrin receptor (CD71). After expressing the fusion protein in E. coli, the gene product was isolated from inclusion bodies and tested for cytotoxicity. RESULTS: This fusion protein inhibited the protein synthesis of three human tumor cell lines derived from a melanoma, a renal carcinoma and a breast carcinoma, with IC50s of 8, 5 and 10 nM, respectively. These values were comparable with those using a similar fusion protein constructed with eosinophil derived neurotoxin (EDN) as the toxic moiety (IC50s of 8, 1.2 and 3 nM, respectively). The slightly lower activities of the human pancreatic RNase-scFv (pancRNase-scFv) with two of the cell lines suggests that fewer molecules are reaching the cytoplasmic compartment, since it was twice as active as EDN-scFv in inhibiting the protein synthesis of a rabbit reticulocyte lysate. CONCLUSION: These results demonstrate that the human pancreatic RNase, which is expected to have a very low immunogenic potential in humans with no inherent toxicity, may be a potent cytotoxin for tumor cells after antibody targeting.

The genomic organization of a human creatine transporter (CRTR) gene located in Xq28.

During the course of a large-scale sequencing project in Xq28, a human creatine transporter (CRTR) gene was discovered. The gene is located approximately 36 kb centromeric to ALD. The gene contains 13 exons and spans about 8.5 kb of genomic DNA. Since the creatine transporter has a prominent function in muscular physiology, it is a candidate gene for Barth syndrome and infantile cardiomyopathy mapped to Xq28.

A gene mutated in X-linked myotubular myopathy defines a new putative tyrosine phosphatase family conserved in yeast.

X-linked recessive myotubular myopathy (MTM1) is characterized by severe hypotonia and generalized muscle weakness, with impaired maturation of muscle fibres. We have restricted the candidate region to 280 kb and characterized two candidate genes using positional cloning strategies. The presence of frameshift or missense mutations (of which two are new mutations) in seven patients proved that one of these genes is indeed implicated in MTM1. The protein encoded by the MTM1 gene is highly conserved in yeast, which is surprising for a muscle specific disease. The protein contains the consensus sequence for the active site of tyrosine phosphatases, a wide class of proteins involved in signal transduction. At least three other genes, one located within 100 kb distal from the MTM1 gene, encode proteins with very high sequence similarities and define, together with the MTM1 gene, a new family of putative tyrosine phosphatases in man.

A 900-kb cosmid contig and 10 new transcripts within the candidate region for myotubular myopathy (MTM1).

The X-linked myotubular myopathy locus (MTM1) has been assigned to the Xq28 region by linkage analysis. By observation of an interstitial deletion in a female patient, the candidate region could be further reduced to a region of 600 kb flanked by the markers DXS304 and DXS497. We describe here cosmid contigs covering a region of 900 kb, including the entire MTM1 candidate region. Cosmids from the region were used to construct an enriched cDNA library from this area. Filter grids carrying this library were then screened by hybridization with whole cosmid clones, with CpG island-containing fragments from linking clones located in the area, and with total exon trap products of cosmid clones from the candidate region. In this analysis, 10 new transcripts were identified and localized precisely within the map. Genes in this area are candidates for MTM1 and a number of other diseases localized by genetic linkage studies to the chromosomal band Xq28.

Isolation, differential splicing and protein expression of a DNase on the human X chromosome.

A systematic search for genes differentially expressed in human tissues resulted in the isolation of a gene encoding a protein with high homology to DNase I. In addition to the recently described cDNA sequence (Parrish et al., 1995) we have isolated a transcript, alternatively spliced in the 5' noncoding region. The gene is located between the QM and the XAP-2 gene in Xq28 and encodes a 302 amino acid protein with 39% identity to human DNase I. Besides a high homology at the nucleotide and amino acid level, most exon-intron boundaries of DNase I and DNase X are identical, indicating that both genes may have evolved from a common ancestor. The predicted function was verified by expression of a recombinant protein in an inducible bacterial system and detection of DNase activity. In contrast to DNase I a 18 kdal amino terminal fragment of the full length 35 kdal protein exhibited DNase activity.

Molecular cloning of tissue-specific transcripts of a transketolase-related gene: implications for the evolution of new vertebrate genes.

As part of a systematic search for differentially expressed genes, we have isolated a novel transketolase-related gene (TKR) (HGMW-approved symbol TKT), located between the green color vision pigment gene (GCP) and the ABP-280 filamin gene (FLN1) in Xq28. Transcripts encoding tissue-specific protein isoforms could be isolated. Comparison with known transketolases (TK) demonstrated a TKR-specific deletion mutating one thiamine binding site. Genomic sequencing of the TKR gene revealed the presence of a pseudoexon as well as the acquisition of a tissue-specific spliced exon compared to TK. Since it has been postulated that the vertebrate genome arose by two cycles of tetraploidization from a cephalochordate genome, this could represent an example of the modulation of the function of a preexisting transketolase gene by gene duplication. Thiamine defiency is closely involved with two neurological disorders, Beriberi and Wernicke-Korsakoff syndromes, and in both of these conditions TK with altered activity are found. We discuss the possible involvement of TKR in explaining the observed variant transketolase forms.

Ordering of 66 STSs along the entire short arm of human chromosome 17 and chromosome assignment of a transcribed sequence (FMR1L2) homologous to FMR1.

Sixty-four PCR-markers previously assigned to the short arm of chromosome 17 and two newly established STSs were localized on a hybrid cell-YAC clone panel. The 66 STSs fell into 23 unique retention patterns, providing a map converting the entire short arm of human chromosome 17 with an average resolution of approximately 1.2 Mb. The combination of radiation-reduced hybrids, somatic cell hybrids and selected YAC clones enabled the precise localization of break-points in two cell hybrids. Since polymorphic STSs from the CEPH as well as the UTAH genetic map were used in this study, a physical link has been generated between these two high resolution genetic maps. FMR1L2, a second FMR1 autosomal homologue has been identified and assigned to a genomic interval between D17S796 and D17S799.

Highly conserved 3' UTR and expression pattern of FXR1 points to a divergent gene regulation of FXR1 and FMR1.

A search for genes with sequence homologies to the FMR1 gene resulted in the isolation of mouse and human homologues of the recently described FXR1 gene. The mouse FXR1 gene shares amino acid identity and similarity of 99.1% and 99.6%, respectively, with the human FXR1 gene and amino acid identify and similarity of 67.3% and 79.5% respectively, with the mouse FMR1 gene. The 3' untranslated region of the FXR1 gene is extremely conserved between human and mouse. The gene structure of FXR1 is very similar to that of FMR1 and both genes probably originate from a common ancestral gene. In contrast to the previously published localization, we mapped the transcribed gene to chromosome region 3q28. An intronless form of the FXR1 gene, either processed functional homologue or pseudogene was localized to 12q12. Northern blot analysis of the human FXR1 gene revealed an expression pattern of a housekeeping gene with stronger expression in muscle. RNA in situ hybridization to sections of mouse embryo and adult tissues has shown that during embryonic development the mouse FXR1 mRNA is expressed in different tissues, most prominent in skeletal muscle, the gonads and distinct regions of the central nervous system, and that the expression is restricted to proliferating cells. While FMR1 is highly expressed in proliferating spermatogonia, FXR1 is highly expressed in postmeiotic spermatids.