Multi-centre phase II clinical trial of yttrium-90 resin microspheres alone in unresectable, chemotherapy refractory colorectal liver metastases.

BACKGROUND: This multi-centre phase II clinical trial is the first prospective evaluation of radioembolisation of patients with colorectal liver metastases (mCRC) who failed previous oxaliplatin- and irinotecan-based systemic chemotherapy regimens. METHODS: Eligible patients had adequate hepatic, haemopoietic and renal function, and an absence of major hepatic vascular anomalies and hepato-pulmonary shunting. Gastroduodenal and right gastric arteries were embolised before hepatic arterial administration of yttrium-90 resin microspheres (median activity, 1.7 GBq; range, 0.9-2.2). RESULTS: Of 50 eligible patients, 38 (76%) had received > or =4 lines of chemotherapy. Most presented with synchronous disease (72%), >4 hepatic metastases (58%), 25-50% replacement of total liver volume (60%) and bilateral spread (70%). Early and intermediate (>48 h) WHO G1-2 adverse events (mostly fever and pain) were observed in 16 and 22% of patients respectively. Two died due to renal failure at 40 days or liver failure at 60 days respectively. By intention-to-treat analysis using Response Evaluation Criteria in Solid Tumours, 1 patient (2%) had a complete response, 11 (22%) partial response, 12 (24%) stable disease, 22 (44%) progressive disease; 4 (8%) were non-evaluable. Median overall survival was 12.6 months (95% CI, 7.0-18.3); 2-year survival was 19.6%. CONCLUSION: Radioembolisation produced meaningful response and disease stabilisation in patients with advanced, unresectable and chemorefractory mCRC.

Automated continuous culture of mammalian cells in suspension.

A system has been developed for the continuous culture of mammalian cells in suspension. The system maintains constant cell concentrations (monitored as the degree of light scattering) over a wide range of previously selected values by automatic additions of known amounts of medium and simultaneous withdrawals of equal volumes of cell suspension.

The transcriptionally competent U2 gene is necessary and sufficient for adenovirus type 12 induction of the fragile site at 17q21-22.

Adenovirus type 12 induces four fragile sites upon infection of human cells. The U2 locus, consisting of up to 20 tandem repeats of a 5.8-kbp monomer, maps at the most sensitive of these sites at 17q21-22. We have previously shown that an artificial U2 locus integrated into the human genome generates a new virus-induced fragile site. To determine which elements within the U2 monomer are responsible for fragility, we constructed loci consisting of tandem repeats of subfragments of the U2 monomer. With this approach, we demonstrate that a transcriptionally competent U2 gene is necessary and sufficient for virus-induced fragility and that no other element within the 5.8-kbp monomer contributes to this effect.

Generation of a new adenovirus type 12-inducible fragile site by insertion of an artificial U2 locus in the human genome.

Infection with adenovirus type 12 (Ad12) induces four fragile sites in the human genome (H.F. Stich, G.L. van Hoosier, and J.J. Trentin, Exp. Cell Res. 34:400-403, 1964; H. zur Hausen, J. Virol. 1:1174-1185, 1967). The major site, at 17q21-22, contains the U2 gene cluster, which is specifically disrupted by infection in at least a percentage of the cells (D.M. Durnam, J.C. Menninger, S.H. Chandler, P.P. Smith, and J.K. McDougall, Mol. Cell. Biol. 8:1863-1867, 1988). For direct assessment of whether the U2 locus is the target of the Ad12 effect, an artificial locus, constructed in vitro and consisting of tandem arrays of the U2 6-kbp monomer, was transfected into human cells. We report that integration of this artificial locus on the p arm of chromosome 13 creates a new Ad12-inducible fragile site.

Reprogramming of telomerase by expression of mutant telomerase RNA template in human cells leads to altered telomeres that correlate with reduced cell viability.

Telomerase synthesizes telomeric DNA by copying the template sequence of its own RNA component. In Tetrahymena thermophila and yeast (G. Yu, J. D. Bradley, L. D. Attardi, and E. H. Blackburn, Nature 344:126-131, 1990; M. McEachern and E. H. Blackburn, Nature 376:403-409, 1995), mutations in the template domain of this RNA result in synthesis of mutant telomeres and in impaired cell growth and survival. We have investigated whether mutant telomerase affects the proliferative potential and viability of immortal human cells. Plasmids encoding mutant or wild-type template RNAs (hTRs) of human telomerase and the neomycin resistance gene were transfected into human cells to generate stable transformants. Expression of mutant hTR resulted in the appearance of mutant telomerase activity and in the synthesis of mutant telomeres. Transformed cells were not visibly affected in their growth and viability when grown as mass populations. However, a reduction in plating efficiency and growth rate and an increase in the number of senescent cells were detected in populations with mutant telomeres by colony-forming assays. These results suggest that the presence of mutant telomerase, even if coexpressed with the wild-type enzyme, can be deleterious to cells, likely as a result of the impaired function of hybrid telomeres.

Induction of hTERT expression and telomerase activity by estrogens in human ovary epithelium cells.

In mammals, molecular mechanisms and factors involved in the tight regulation of telomerase expression and activity are still largely undefined. In this study, we provide evidence for a role of estrogens and their receptors in the transcriptional regulation of hTERT, the catalytic subunit of human telomerase and, consequently, in the activation of the enzyme. Through a computer analysis of the hTERT 5'-flanking sequences, we identified a putative estrogen response element (ERE) which was capable of binding in vitro human estrogen receptor alpha (ERalpha). In vivo DNA footprinting revealed specific modifications of the ERE region in ERalpha-positive but not ERalpha-negative cells upon treatment with 17beta-estradiol (E2), indicative of estrogen-dependent chromatin remodelling. In the presence of E2, transient expression of ERalpha but not ERbeta remarkably increased hTERT promoter activity, and mutation of the ERE significantly reduced this effect. No telomerase activity was detected in human ovary epithelial cells grown in the absence of E2, but the addition of the hormone induced the enzyme within 3 h of treatment. The expression of hTERT mRNA and protein was induced in parallel with enzymatic activity. This prompt estrogen modulation of telomerase activity substantiates estrogen-dependent transcriptional regulation of the hTERT gene. The identification of hTERT as a target of estrogens represents a novel finding which advances the understanding of telomerase regulation in hormone-dependent cells and has implications for a potential role of hormones in their senescence and malignant conversion.

Construction of a recombinant adenovirus for efficient delivery of the I-SceI yeast endonuclease to human cells and its application in the in vivo cleavage of chromosomes to expose new potential telomeres.

We have constructed a replication-defective adenovirus vector encoding the yeast I- Sce I endonuclease under the control of the murine cytomegalovirus immediate-early gene promoter (AdM Sce I) for efficient delivery of this enzyme to mammalian cells. We present evidence of AdM Sce I-mediated I- Sce I protein expression and cleavage activity in replication-permissive 293 cells, and of cleavage of chromosomes in vivo in both 293 cells and in non-permissive human cells. We have exploited this system for the generation of chromosomes capped by artificial telomeric sequences in cells with integrated plasmids containing telomeric DNA arrays adjacent to an I- Sce I recognition site. The properties of the AdM Sce I virus described here make it a useful tool for studying biological processes involving induction of DNA breaks, recombination and gene targeting in cells grown in culture and in vivo.

A multicentric phase II clinical trial on intra-arterial hepatic radiotherapy with 90yttrium SIR-spheres in unresectable, colorectal liver metastases refractory to i.v. chemotherapy: preliminary results on toxicity and response rates.

BACKGROUND: In patients locally progressing after two lines of chemotherapy, some locoregional approaches showed encouraging results in terms of local control of disease. The aim of our study was to evaluate toxicity, clinical response and quality of life in 48 patients with unresectable colorectal liver metastases submitted to selective internal radiotherapy (SIRT). MATERIALS AND METHODS: Up to now 35 patients with unresectable colorectal liver metastases, refractory to two lines of chemotherapy, underwent intra-arterial infusion of resin microspheres with yttrium-90 (SIR-spheres). Pre-treatment evaluation included a CT scan, blood tests, a PET scan and arteriography of celiac trunk, hepatic and superior mesenteric artery; extrahepatic uptakes and pulmonary shunts more than 10% were excluded by a Scinti-scan. The gastroduodenal artery was embolized before the SIR-spheres injection. Other exclusion criteria were liver dysfunction and anatomical vascular anomalies. The clinical response was evaluated by CT-scan following the RECIST criteria. Median follow-up was 4 months. RESULTS: Median number of metastases was 4 (range, 1-15), 38% of cases presenting hepatic involvement < 25%. The median SIRT dose delivered was 1.7 GBq. Median pulmonary shunt was 6%. No operative mortality occurred; early toxicity (within 48 hours) was 20.6%, shown as fever, acute pain and leucocytosis. The late toxicity was 24.1% with chronic pain, jaundice and nausea being the most frequent. All the toxic events were graded 2 or 3 according to the WHO scale. Preliminary results were available in terms of clinical response after 6 weeks: 12.5% had a partial response, 75% a stable disease, while progression of disease, was observed in 12.5% of the patients. CONCLUSION: SIRT is a safe treatment in terms of acute and late toxicity. Intra-arterial microspheres could represent a good therapeutic option for patients with progressing liver metastases only, after two lines of systemic chemotherapy.

Development of retinoblastoma in the absence of telomerase activity.

BACKGROUND: The length and stability of telomeres (essential functional structures at the end of eukaryotic chromosomes) have been implicated in the control of cell lifespan. Most somatic cells lack telomerase, the enzyme that synthesizes telomeric DNA, and their telomeres shorten with cell division. Cells immortalized in vitro, on the other hand, express telomerase and maintain their telomeres. Telomerase activity has also been detected in the large majority of tumors from a variety of cancers. These observations have suggested that telomere maintenance is required for unlimited cell proliferation and that telomerase is a marker for cell immortality in vitro and in vivo. PURPOSE: We investigated whether telomerase is activated during the development of retinoblastoma. This is a childhood eye cancer associated with a limited number of mutations in an embryonic tissue and thus likely to develop in cells that have long telomeres. The ease of detection of retinoblastoma makes it possible to screen relatively small tumors before extensive proliferation of the malignant cells. METHODS: We measured telomerase activity in 34 samples of retinoblastoma, four retinoblastoma-derived cell lines, and six cell lines derived from other cancers. Only three of the cell lines from other cancers expressed the retinoblastoma protein. Telomerase activity was assayed by a polymerase chain reaction protocol in extracts prepared from tumors or cell lines. The level of enzyme activity in cell extracts was quantified at several protein concentrations and expressed relative to that in a positive control, after normalization for the amount of protein. Telomere length was measured by Southern blot hybridization of genomic DNA with a telomere-specific probe. Average values of telomere length in telomerase-positive and telomerase-negative tumors and in cell lines were compared by two-sided, two-sample Student's t test. RESULTS: No telomerase activity was detected in 17 (50%) of 34 retinoblastomas. Assays of cell lines derived from other cancers revealed no association between the presence or the level of the enzyme activity and the expression of the retinoblastoma protein. Telomeres were significantly longer in telomerase-negative tumors than in telomerase-positive tumors (P = .0008). CONCLUSIONS: Our results indicate that retinoblastoma can develop when telomeres are still relatively long and in the absence of telomerase. Telomerase activity associated with short telomeres is, however, observed in 50% of the retinoblastomas and in retinoblastoma-derived cell lines. IMPLICATIONS: Telomerase may not be a marker for acquisition of the malignant phenotype in the case of tumors that are derived from cells with long telomeres and that are associated with a low number of mutations.

Telomerase activity associated with acquisition of malignancy in human colorectal cancer.

Shortening of telomeres may contribute to the control of the proliferative capacity of normal cells, and telomerase, the enzyme that elongates telomeric DNA, may be essential for unlimited cell proliferation. We have shown previously that telomerase activity is present in human cells immortalized in vitro and in metastatic ovarian carcinoma cells but is undetectable in normal cultured cells or normal tissues. We have determined the temporal pattern of telomerase activity during colorectal carcinogenesis in man. We report that telomerase activity is associated with acquisition of malignancy as it is detectable in colorectal carcinoma but not in adenomatous polyps. Mutations leading to reactivation or upregulation of the enzyme may represent an additional required event in the multistep development of colorectal cancer.

Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues.

Telomerase activity has been detected in many human immortal cells lines and in tumor tissues, whereas it is generally absent from primary cell strains and from many tumor adjacent tissue samples. With the recently cloned human telomerase RNA (hTR), we used Northern analysis to follow the levels of hTR in primary, precrisis, and immortalized cells. It was surprising that the amount of hTR was high in cell strains that lacked telomerase activity, and the levels did not parallel the increases in telomerase activity, which accompanies immortalization. In addition, although the hTR levels were somewhat higher in tumor samples compared to nontumor tissues, the level of hTR in a variety of different human tumors did not predict the level of telomerase activity in the tumor. Thus, whereas hTR was detected in all samples that have telomerase activity, the presence of the RNA was not a good predictor of the presence or amount of telomerase activity.

Induction of genomic instability in SV40 transformed human cells: sufficiency of the N-terminal 147 amino acids of large T antigen and role of pRB and p53.

Genomic instability is an early event in the transformation of human cells by SV40 and may contribute, as a mutagenic process, to the generation of the rare cells which survive crisis and yield immortal populations. We have previously reported that expression of large T antigen is responsible for induction of chromosome aberrations and aneuploidy. In the present study we have demonstrated that the amino terminal 147 amino acids of the protein are as proficient as full length T antigen for this destabilization of the cell genome. Analysis of mutants within this region indicated that T antigens defective for binding to pRB or lacking the first 127 amino acids are significantly reduced in their ability to induce aneuploidy and/or aberrations, whereas a cytoplasmic T antigen is less severely impaired. In addition, we have shown that binding of T antigen to p53 is dispensable for genome destabilization but may be required for continued proliferation of genetically aberrant cells.

Expression of mutant telomerase in immortal telomerase-negative human cells results in cell cycle deregulation, nuclear and chromosomal abnormalities and rapid loss of viability.

We have reconstituted wild type or mutant telomerase activity in two human cell lines that lack constitutive expression of both core subunits of the enzyme and maintain telomeres by a telomerase-independent mechanism (ALT cells). Wild type telomerase RNA and four telomerase RNAs with single point mutations in their template domain were used to express enzymes specifying different telomeric DNA sequences. Expression of wild type telomerase for up to 32 days had no detectable effect on cell growth or viability. In contrast, cells expressing mutant telomerases had slower growth rate, abnormal cell cycle and reduced viability. Dramatically aberrant nuclei, typical of cells undergoing mitotic catastrophe, and large numbers of fused chromosomes were also characteristic of these populations. Notably, all phenotypes were apparent within the first few cell divisions after expression of the enzymes. Unlike wild type, mutant telomerase activity was progressively selected against with cell culturing, and this correlated with the disappearance of cells with aberrant phenotypes. Our results suggest that even very limited synthesis of mutated sequences can affect telomere structure in human cells, and that the toxicity of mutant telomerases is due to telomere malfunction.

Telomerase activity in normal and malignant murine tissues.

Telomere shortening may contribute to the limited lifespan of somatic cells and telomerase, the enzyme that elongates telomeric DNA and maintains telomere length, may be essential for unlimited cell proliferation in vivo and in vitro. Telomerase is not expressed in most human somatic cells but is a nearly ubiquitous tumour marker, being activated in malignant cells from many cancers. Inhibition of telomerase may lead to telomere shortening and eventually limit the proliferative capacity of malignant cells and hence be of therapeutic value. With the intent of characterizing an animal model for inhibition studies, we investigated telomerase activity during mammary tumorigenesis in transgenic mice overexpressing the neu gene. We detected activity in primary mammary tumours and lung metastases but also in normal mammary glands and other organs. Activity was elevated in tumors versus normal tissues and was enhanced by short-term culturing of normal cells. Telomerase activity was also present in somatic tissues from the non-transgenic parental strain and the outbred Mus spretus strain. As we recently detected telomerase activity in normal human hemopoietic tissues, mouse models of tumorigenesis may provide useful experimental systems for assessing the outcome of in vivo inhibition of telomerase in both malignant and normal cells.

Up-regulation of CIR1/CROC1 expression upon cell immortalization and in tumor-derived human cell lines.

Acquisition of the immortal phenotype by tumor cells represents an essential and potentially rate-limiting step in tumorigenesis. To identify changes in gene expression that are associated with the early stages of cell immortalization, we compared genetically matched pairs of pre-immortal and immortal human cell clones by mRNA differential display. Two transcripts, denoted CIR1 and CIR2, were identified which were up-regulated in immortal cells. Sequence analysis revealed CIR1 to be identical to the recently cloned CROC1/UEV-1 gene, whereas CIR2 corresponds to an as yet uncharacterized 1.2 kb mRNA. A 5-6-fold elevation in CIR1/CROC1 expression and a 2-3-fold elevation in CIR2 expression were observed in SV40-transformed human embryonic kidney cells immediately following proliferative crisis, suggesting a potential role for these genes in immortalization. Expression of CIR1/CROC1 was found to be elevated also in a variety of immortal human tumor-derived cell lines, as compared to their normal tissue counterparts. These results are compatible with induction of CIR1/CROC1 being an early event in the acquisition of immortality and with a role for this gene in the immortal phenotype of tumor cells.

Purification and characterization of an endonuclease from calf thymus acting on irradiated DNA.

An endonuclease acting on DNA exposed to ultraviolet light or gamma-rays has been extensively purified from calf thymus. The enzyme has a pH optimum at pH 7.0-7.5, acts with equal efficiency in the presence of EDTA or divalent cations (Mg-2+ or Ca-2+), is inhibited by NaCl and tRNA and is inactivated by incubation at 50 degrees C. Its molecular weight, determined by Sephadex chromatography or sodium dodecylsulfate gel electrophoresis, is approx. 30 000. The enzyme catalyzes the formation of breaks with 5'-phosphate termini in double-stranded DNA irradiated with ultraviolet or gamma-rays. It does not act on unirradiated DNA or denatured DNA. Since in all these properties the enzymatic activity on ultraviolet- and gamma-irradiated DNA behaved similarly and since the two activities cochromatographed in all systems used during purification, we conclude that they are associated with the same protein. The site of action of the enzyme in ultraviolet-irradiated DNA is a photoproduct other than pyrimidine dimers. Such a photoproduct can also be induced by irradiation of the DNA in vivo, i.e. within the cells.

Tn5 mutagenesis of the transforming genes of human adenovirus type 5.

We have cloned the entire human adenovirus type 5 (Ad5) genome into the pBR322 plasmid in two segments: the BamHI-A fragment (21 kb) and the BamHI-B fragment (15 kb). We have also generated a series of clones with smaller Ad5 DNA inserts, all containing the left-end of the viral genome. One such clone, pXCl, containing the left 16% of the Ad5 DNA molecule, has been shown to transform rodent cells by DNA transfection. We have used the transposable element Tn5 as an insertion mutator to isolate pXCl mutants containing Tn5 inserted at a large number of sites. By assaying transforming activity of selected pXC::Tn5 plasmids we have identified Ad5 sequences which are essential for DNA-mediated transformation. Our results with these mutants and with a plasmid pCD1, containing a deletion within the Ad5-transforming region, indicate that sequences present in both early region 1a and the N-terminal region of early region 1b are essential for DNA-mediated transformation.

A survey of telomerase activity in human cancer.

Research on the association of the ribonucleoprotein enzyme, telomerase, with human cancer has expanded rapidly in recent years. Essentially all major types of cancer have been screened and the presence of telomerase activity has been detected in the vast majority of cases. In this article we provide a summary, in table form, of the current data.

The responsiveness of human papillomavirus upstream regulatory regions to herpes simplex virus immediate early proteins.

We have tested the responsiveness of the upstream regulatory regions (URR) of human papillomaviruses (HPV) to transactivation by the herpes simplex virus type 1 (HSV-1) immediate early proteins ICP4 (Vmw175) and ICP0 (Vmw110), using recombinant plasmids which contain URRs of HPV 1, 11 or 16 upstream of an SV40-promoted CAT gene. Transfection of the HPV-CAT plasmids into mouse cells expressing a temperature-sensitive ICP4 protein enabled us to demonstrate that ICP4 enhances transcription of the HPV 1 and 16 URRs, but not of the HPV 11 URR. Cotransfection of the HPV CAT plasmids with a plasmid encoding ICP0 indicated that only the URR of HPV 16 responded to this protein, and that the response was host cell dependent.

Inhibition of cell-proliferation by an adenovirus vector expressing the human wild type-p53 protein.

We have developed human adenovirus 5 (Ad5) vectors expressing the wild type human p53 protein or a mutant p53 form under the control of the human cytomegalovirus immediate early gene promoter. Human cells infected with these vectors expressed high levels of p53, accumulating 20-40 fold more protein than found in normal human fibroblasts. The ability of the vectors to affect proliferation of cells in culture was assessed by measuring cell DNA synthesis and colony forming ability after infection with viruses. When the p53 deficient ovarian carcinoma cell line, SKOV-3, was infected with Adp53wt expressing the wild type (wt) p53 protein, a significant inhibition of cellular DNA synthesis was observed, relative to cells infected with Adp53m expressing mutant p53, or a control virus, AdLacZ, expressing bacterial beta-galactosidase. Inhibition was dependent on multiplicity of infection, with no significant effect below 5 pfu/cell, and maximal effect between 25 and 100 PFU/cell which resulted in approximately 95% inhibition of SKOV-3 cell DNA synthesis relative to mock infected cells. Infection of normal human fibroblasts with Adp53wt also inhibited DNA synthesis but to a significantly lesser degree. SKOV-3 cell survival, assayed by ability to form colonies, was reduced at least 10 fold after infection with Adp53wt compared to colony forming ability of cells after infection with either AdLacZ or Adp53m. The results of these studies indicate that p53 expressed by Ad vectors can inhibit proliferation in culture of p53 negative cells by at least 95%, and suggest that such vectors might similarly inhibit the proliferation of tumor cells in vivo.