High level of telomerase RNA gene expression is associated with chromatin modification, the ALT phenotype and poor prognosis in liposarcoma.

Telomere length is maintained by two known mechanisms, activation of telomerase or alternative lengthening of telomeres (ALT). The ALT pathway is more commonly activated in tumours of mesenchymal origin, although the mechanisms involved in the decision of a cell to activate either telomerase or ALT are unknown at present and no molecular markers exist to define the ALT phenotype. We have previously shown an association between chromatin remodelling, telomerase gene expression and ALT in cell line models. Here, we evaluate these findings and investigate their prognostic significance in a panel of liposarcoma tissue samples to understand the biology underlying the ALT phenotype. Liposarcoma samples were split into three groups: telomerase positive (Tel+); ALT positive; ALT-/Tel-. Differences in telomerase gene expression were evident between the groups with increased expression of hTR in ALT and Tel+ compared to ALT-/Tel- samples and increased hTERT in Tel+ samples only. Investigation of a small panel of chromatin modifications revealed significantly increased binding of acetyl H3 in association with hTR expression. We confirm that the presence of the ALT phenotype is associated with poor prognosis and in addition, for the first time, we show a direct association between hTR expression and poor prognosis in liposarcoma patients.

Hypoxic regulation of telomerase gene expression by transcriptional and post-transcriptional mechanisms.

Basal telomerase activity is dependent on expression of the hTERT and hTR genes and upregulation of telomerase gene expression is associated with tumour development. It is therefore possible that signal transduction pathways involved in tumour development and features of the tumour environment itself may influence telomerase gene regulation. The majority of solid tumours contain regions of hypoxia and it has recently been demonstrated that hypoxia can increase telomerase activity by mechanisms that are still poorly defined. Here, we show that hypoxia induces the transcriptional activity of both hTR and hTERT gene promoters. While endogenous hTR expression is regulated at the transcriptional level, hTERT is subject to regulation by alternative splicing under hypoxic conditions, which involves a switch in the splice pattern in favour of the active variant. Furthermore, analysis of the chromatin landscape of the telomerase promoters reveals dynamic recruitment of a transcriptional complex involving the hypoxia-inducible factor-1 transcription factor, p300, RNA polymerase II and TFIIB, to both promoters during hypoxia, which traffics along and remains associated with the hTERT gene as transcription proceeds. These studies show that hTERT and hTR are subject to similar controls under hypoxia and highlight the rapid and dynamic regulation of the telomerase genes in vivo.

Dysregulated expression of the major telomerase components in leukaemic stem cells.

Telomere loss is rapid during the progression of chronic myeloid leukaemia (CML) and correlates with prognosis. We therefore sought to measure expression of the major telomerase components (hTR and hTERT) in CD34+ cells from CML patients and normal controls, to determine if their altered expression may contribute to telomere attrition in vivo. High-purity (median 94.1%) BCR-ABL+ CD34+ cells from CML (n=16) and non-CML (n=14) patients were used. CML samples had a small increase in telomerase activity (TA) compared to normal samples (approximately 1.5-fold, P=0.004), which was inversely correlated with the percentage of G0 cells (P=0.02) suggesting TA may not be elevated on a cell-to-cell basis in CML. Consistent with this, hTERT mRNA expression was not significantly elevated; however, altered mRNA splicing appeared to play a significant role in determining overall full length, functional hTERT levels. Interestingly, Q-RT-PCR for hTR demonstrated a mean five-fold reduction in levels in the chronic phase (CP) CML samples (P=0.002), raising the possibility that telomere homeostasis is disrupted in CML. In summary, the molecular events regulating telomerase gene expression and telomere maintenance during the CP of CML may influence the disease progression observed in these patients.

Lack of telomerase RNA gene hTERC expression in alternative lengthening of telomeres cells is associated with methylation of the hTERC promoter.

The immortal phenotype of most human cancers is attributable to telomerase expression. However, a number of immortal cell lines and tumors achieve telomere maintenance in the absence of telomerase via alternative mechanisms known as ALT (alternative lengthening of telomeres). Here we show that the promoter of the telomerase RNA gene (hTERC) is methylated in three of five ALT cell lines and is associated with a total absence of hTERC expression in the three lines. Treatment with 5-azacytidine in combination with trichostatin A resulted in partial demethylation of the hTERC promoter and expression of the gene. Partial methylation was detected in tumors (5%) and in immortal cell lines (27%). Cell lines with partial methylation express hTERC. Only in ALT cell lines does there appear to be a strong correlation between hTERC promoter hypermethylation and lack of hTERC expression.

Mapping of the gene for the human telomerase reverse transcriptase, hTERT, to chromosome 5p15.33 by fluorescence in situ hybridization.

Telomerase, the enzyme that maintains the ends of chromosomes, is absent from the majority of somatic cells but is present and active in most tumours. The gene for the reverse transcriptase component of telomerase (hTERT) has recently been identified. A cDNA clone of this gene was used as a probe to identify three genomic bacterial artificial chromosome (BAC) clones, one of which was used as a probe to map hTERT by fluorescence in situ hybridization (FISH) to chromosome 5p15.33. This BAC probe was further used to look at copy number of the hTERT region in immortal cell lines. We found that 10/15 immortal cell lines had a modal copy number of 3 or more per cell, with one cell line (CaSki) having a modal copy number of 11. This suggests that increases in copy number of the hTERT gene region do occur, and may well be one route to upregulating telomerase levels in tumour cells. 5p15 gains and amplifications have been documented for various tumour types, including non-small cell lung carcinoma, squamous cell carcinoma of head and neck, and uterine cervix cancer, making hTERT a potential target.

Activation of telomerase rna gene promoter activity by NF-Y, Sp1, and the retinoblastoma protein and repression by Sp3.

Expression of the human telomerase RNA component gene, hTERC is essential for telomerase activity. The hTERC gene is expressed during embryogenesis and then downregulated during normal development, leaving most adult somatic cells devoid of hTERC expression. During oncogenesis, however, hTERC is re-expressed consequently contributing to the unrestricted proliferative capacity of many human cancers. Thus the identification of the molecular basis for the regulation of the telomerase RNA component gene in normal cells and its deregulation in cancer cells is of immediate interest. We have previously cloned the hTERC promoter and in this study have identified several transcription factors that modulate the expression of hTERC. We demonstrate that NF-Y binding to the CCAAT region of the hTERC promoter is essential for promoter activity. Sp1 and the retinoblastoma protein (pRb) are activators of the hTERC promoter and Sp3 is a potent repressor. These factors appear to act in a species-specific manner. Whereas Sp1 and Sp3 act on the human, bovine, and mouse TERC promoters, pRb activates only the human and bovine promoter, and NF-Y is only essential for the human TERC gene.

Cloning and characterization of human and mouse telomerase RNA gene promoter sequences.

Variation in telomerase activity is correlated with cellular senescence and tumour progression. However, although the enzymatic activity of telomerase has been well studied, very little is known about how expression of telomerase genes is regulated in mammalian cells. We have therefore cloned the promoter regions of the human (hTR), and mouse, (terc), telomerase RNA genes in order to identify the regulatory elements controlling telomerase RNA gene transcription. 1.76 kb encompassing the hTR gene promoter region was sequenced, as was 4 kb encompassing the terc promoter. No significant sequence similarity could be detected in comparisons between human and mouse 5'-regions, flanking the transcribed sequences. However, both the human and mouse telomerase RNA genes are within CpG islands and may therefore be under the regulation of DNA methylation. Transient expression of hTR-reporter gene constructs in HeLa and GM847 cells identified the elements responsible for promoter activity are contained in a 231 bp region upstream of the transcriptional start site. Transient expression of terc-reporter gene constructs in Swiss3T3 and A9 cells identified the elements responsible for promoter activity are contained in a 73 bp region upstream of the transcriptional start site. These studies have implications for novel transcription targeted cancer therapies.

Amplification, increased dosage and in situ expression of the telomerase RNA gene in human cancer.

Telomere length is maintained by the enzyme, telomerase, which has been linked to cellular immortality and tumour progression. However, the reasons for the high levels of telomerase found in human tumours are unknown. We have mapped the human telomerase RNA gene, (hTR), to chromosome 3q26.3 and show the hTR gene to be amplified in four carcinomas, (2/33 cervix, 1/31 head and neck, 1/9 lung). In addition, increased copy numbers of the hTR locus was also observed in 97% of tumours. By in situ hybridisation, the histological distribution of high levels of hTR expression could be demonstrated in a lung tumour and its metastasis with hTR amplification. These results are the first report of genetic alterations involving a known component of telomerase in human cancer. Indeed, it is also the first report of the amplification of a specific locus within the chromosome 3q region frequently subject to copy number gains in human tumours. In addition, we also show for the first time the histological distribution of the RNA component of telomerase in human tumours.

Identification of genetic changes associated with drug resistance by reverse in situ hybridization.

The molecular cytogenetic techniques of comparative genomic hybridization (CGH) and reverse in situ hybridization (REVISH) allow the entire genomes of tumours to be screened for genetic changes without the requirement for specific probes or markers. In order to define the ability of REVISH to detect and map regions of amplification associated with drug resistance, we investigated a panel of cell lines selected for resistance to doxorubicin and intrinsic sensitivity to topoisomerase II-inhibitory drugs. We have defined a modified REVISH protocol, which involves double hybridizations with genomic DNA from the test cell lines and chromosome-specific whole chromosome paints to identify the chromosomes to which the amplicons localize. Sites of amplification are then mapped by fractional length measurements (Flpter), using published genome databases. Our findings show that amplification of the topoisomerase II alpha gene is readily detected and mapped, as is amplification of the MDR and MRP loci. Interestingly, REVISH detected a new amplicon in the doxorubicin-resistant lung cancer cell line, GLC4-ADR, which mapped to chromosome 1q. REVISH is therefore ideally suited to characterize genetic changes specific for drug resistance within a background of genetic anomalies associated with tumour progression.

Selection of a subpopulation with fewer DNA topoisomerase II alpha gene copies in a doxorubicin-resistant cell line panel.

A panel of doxorubicin-resistant sublines of the human small-cell lung carcinoma cell line GLC4 displays decreasing DNA topoisomerase II alpha (TopoII alpha) mRNA levels with increasing resistance. In the present study we describe how this decrease may be regulated. No significant differences in TopoII alpha mRNA stability or gene arrangement were found, using mRNA slot-blotting and Southern blotting, in the most resistant cell line compared with the parental cell line. To investigate if TopoII alpha gene copy loss contributed to the mRNA decrease, fluorescence in situ hybridisation using a TopoII alpha-specific probe was performed. During doxorubicin resistance development, the composition of the population in each cell line shifted with increasing resistance, from a population in which most cells contain three TopoII alpha gene copies (GLC4) to a population in which most cells contain only two copies. A partial revertant of the most resistant cell line displayed a shift back to the original situation. We conclude that the TopoII alpha gene copy number decrease per cell line is in good agreement with the decreased TopoII alpha mRNA and protein levels, and TopoII activity levels in these cell lines which were described previously.

Characterization of extensive genetic alterations in ductal carcinoma in situ by fluorescence in situ hybridization and molecular analysis.

BACKGROUND: The molecular genetic analysis of invasive breast cancer has identified breast cancer as a genetically complex disease. Ductal carcinoma in situ (DCIS) is thought to represent a preinvasive step in breast cancer progression, yet we know little about its biologic behavior or the genetic alterations present. Because of the increasing diagnosis of DCIS by mammography screening and the debate over how DCIS should be managed, there is a clear need to define the molecular events underlying the development of DCIS. PURPOSE: Our purpose was to identify patterns of genetic alterations in DCIS. METHODS: A group of 30 formalin-fixed, paraffin-embedded blocks of tissue collected from 1987 through 1989 from 21 patients with DCIS was studied. Chromosomal imbalances were determined by interphase cytogenetic analysis using the fluorescence in situ hybridization (FISH) technique. DNA probes were used that recognize chromosome-specific repetitive sequence loci at the centromeres of chromosomes 1, 3, 4, 6, 7, 8, 9, 10, 11, 16, 17, and 18. FISH was also used to detect ERBB2 gene amplification in DCIS. To complement the FISH studies, microsatellite analysis of markers near the BRCA1 region of chromosome 17 was done on tissue microdissected from multiple areas of DCIS. Chromosomal imbalances were determined by comparisons of chromosomal indices (total number of hybridization spots per total number of nuclei counted) of normal and DCIS tissue, using the two-sided Mann-Whitney test. RESULTS: Using FISH, we have identified patterns of DNA loss and gain of certain chromosome-specific centromeric markers in DCIS. We observed frequent gains of markers on chromosomes 3, 10, and 17 as well as loss of chromosome 18-specific centromeric sequences. ERBB2 gene amplification was detected in tumors from four of 15 patients studied and was clearly limited to the tumor cells within the ducts. Because of the availability of topologically distinct regions of tumors from individuals, we were able to show that paired tumor specimens from individuals share genetic alterations and also have unique ones, suggesting clonal diversity within tumors. The combination of FISH and microsatellite analyses suggested that alterations in chromosome 17 may be quite complex; three of five patients whose samples were analyzed had allelic imbalance at markers on the long arm of chromosome 17. CONCLUSIONS: FISH and microsatellite analyses are useful in detecting extensive genetic alterations in DCIS. Examinations of DCIS tissue using these techniques have identified chromosomes 1, 3, 10, 16, 17, and 18 as candidate sites worthy of immediate study. IMPLICATIONS: This approach may give direction to future research aimed at precisely mapping loci altered in DCIS and help in understanding the biologic events associated with tumor progression or recurrence.

Familial benign hypercalcaemia: a possible abnormality in calcium transport by erythrocytes.

The fundamental biochemical abnormality in familial benign hypercalcaemia (FBH) (familial hypocalciuric hypercalcaemia) is unknown. It seemed possible that, since the kidneys and the parathyroid glands are insensitive to the high extracellular calcium levels, a general disorder of the regulation of the calcium pump on the plasma membrane is present. We obtained evidence suggesting that active calcium efflux by erythrocytes from patients with FBH (85.7 +/- 4.5 mumol 1(-1) min-1) is higher (P less than 0.005) than that by erythrocytes from control subjects (78.6 +/- 4.1 mumol 1(-1) min-1) or from patients with primary hyperparathyroidism (77.5 +/- 5.2 mumol 1(-1) min-1, P less than 0.05). Calcium influx into erythrocytes was normal in FBH and in primary hyperparathyroidism.