Biomedical subjects

A M Burger

Publications and source records attributed to A M Burger.

At least 19 recordsLinked to original sources

PubMed2005

Hypoxia-regulated glucose transporter Glut-1 may influence chemosensitivity to some alkylating agents: results of EORTC (First Translational Award) study of the relevance of tumour hypoxia to the outcome of chemotherapy in human tumour-derived xenografts.

Tumour hypoxia confers poor prognosis in a wide range of solid tumours, due to an increased malignancy, increased likelihood of metastasis and treatment resistance. Poorly oxygenated tumours are resistant to both radiation therapy and chemotherapy. However, although the link between radiation therapy and hypoxia is well established in a range of clinical studies, evidence of its influence on chemotherapy response is lacking. In this study, a panel of human tumour-derived xenografts that have been characterised previously for in vivo response to a large series of anti-cancer agents, and have been found to show chemosensitivities that correlate strongly with the parent tumour, were used to address this issue. Immunohistochemistry was carried out on formalin-fixed, paraffin-embedded sections of xenograft samples to detect expression of the intrinsic hypoxia marker Glut-1 and adducts of the bioreductive hypoxia marker pimonidazole. Glut-1 scores correlated significantly with T/C values for CCNU sensitivity (r = 0.439, P = 0.036, n = 23) and showed a borderline significant correlation with dacarbazine T/C (r = 0.405, P = 0.076, n = 20). However, there was no correlation between both Glut-1 and pimonidazole scores and T/C obtained for the bioreductive drug mitomycin C. The use of human tumour-derived xenografts offers a potentially useful way of using archival material to determine the influence of hypoxia and other tumour-microenvironmental factors on chemosensitivity without the direct use of human subjects.

Animals↗

PubMed2004

Clonogenic assay with established human tumour xenografts: correlation of in vitro to in vivo activity as a basis for anticancer drug discovery.

Pluripotent cells can be grown in clonogenic assays. The tumour stem-cell fraction, which accounts for <0.4% of the total cells, and which is considered the most relevant cell type in the development of metastases and recurrences, is able to divide and to form colonies in a semisolid matrix (agar or methylcellulose). Major applications of the tumour clonogenic assay (TCA) are chemosensitivity testing of tumours and xenografts, and for assessments within drug discovery programmes. Of critical relevance for the usefulness of the TCA is whether it can predict sensitivity or resistance towards clinically used agents. When we compared the response of human tumours established as xenografts in nude mice in the TCA in vitro to that of the clinical response, 62% of the comparisons for drug sensitivity, and 92% of the comparisons for drug resistance were correct. The same percentage of true/false observations was found when tumours were tested after serial passage in nude mice in the TCA in vitro and their response compared to in vivo activity in corresponding xenografts (60% and 90%, respectively). The highest correct predictive values were, however, found when the clinical response of tumours was compared to their explants established in the nude mouse and treated in vivo. Of 80 comparisons performed, we observed a correct prediction for tumour resistance in 97% and for tumour sensitivity in 90%. In our opinion, the TCA with established human tumour xenografts has an important role in current drug discovery strategies. We therefore included the TCA as secondary assay in our approach to anticancer drug discovery and found that a number of novel agents were active; these are now in advanced preclinical development or clinical trials. Thus, the tumour clonogenic assay has proven predictive value in the chemosensitivity testing of standard and experimental anticancer drugs.

Animals↗

PubMed2003

The RING-H2 protein RNF11 is overexpressed in breast cancer and is a target of Smurf2 E3 ligase.

The breast cancer-associated T2A10 clone was originally isolated from a cDNA library enriched for tumour messenger ribonucleic acids. Our survey of 125 microarrayed primary tumour tissues using affinity purified polyclonal antibodies has revealed that corresponding protein is overexpressed in invasive breast cancer and is weakly expressed in kidney and prostate tumours. Now known as RNF11, the gene encodes a RING-H2 domain and a PY motif, both of which mediate protein-protein interactions. In particular, the PPPPY sequence of RNF11 PY motif is identical to that of Smad7, which has been shown to bind to WW domains of Smurf2, an E3 ubiquitin ligase that mediates the ubiquitination and degradation of the TGFbeta receptor complex. Using various mutants of RNF11 in GST pulldown and immunoprecipitation assays, we found that RNF11 interacts with Smurf2 through the PY motif, leading to ubiquitination of both proteins. Smurf2 plays an active role in the repression of TGFbeta signalling, and our data indicate that overexpression of RNF11, through its interaction with Smurf2, can restore TGFbeta responsiveness in transfected cells.

Breast Neoplasms↗

PubMed2003

5-Fluorouracil in vesicular phospholipid gels for anticancer treatment: entrapment and release properties.

Vesicular phospholipid gels (VPG), i.e. highly concentrated liposomal dispersions, are suitable for entrapping substances such as anticancer drugs with particular high encapsulation efficiencies (EE). We prepared different formulations of VPG with 30% (w/w) lipid containing 5-fluorouracil (5-FU) by high pressure homogenization and analysed their EE and drug release. Using mixtures of hydrogenated soy phosphatidylcholine and cholesterol with molar ratios ranging from 55/45 to 75/25, a decreasing amount of cholesterol correlated with an increasing EE, which is probably due to a reduced amount of smaller vesicles and number of lamellae. Using a 5-FU solution of pH 8.6 for VPG preparation, an EE of approximately 40% was found after redispersion of the gel to a liposomal dispersion and separation of free drug from liposomal drug by size exclusion chromatography. The reduced EE for preparations with lower pH values was attributed to a fast initial drug release due to the increased drug lipophilicity below the pK(a) value of 8. After redispersion of a VPG of pH 8.0, an initially faster release of about a third of the entrapped drug was found during the first 20 min, followed by stable entrapment over many hours. The rapid initial release may be due to the portion of liposomes smaller than 40 nm in diameter, determined by photon correlation spectroscopy. Cryo electron microscopic pictures show a lentil-like shape of these small liposomes. The membrane defects on the edges are probably the reason for the very high initial drug release rate. The half-life time of the release of 5-FU from intact FU-VPG at both pH 7.4 and 8.0 was found to be in the order of 4-5 h and the kinetics are typical for matrix-controlled drug diffusion. The in vitro data of 5-FU loaded VPG suggest their applicability as implants with controlled release properties or, after redispersion, as intravenously injected liposomal formulations.

Antimetabolites, Antineoplastic↗

PubMed2002

Change in pharmacokinetic and pharmacodynamic behavior of gemcitabine in human tumor xenografts upon entrapment in vesicular phospholipid gels.

PURPOSE: The in vivo pharmacokinetics (PK), biodistribution and antitumor activity of a new liposomal formulation of gemcitabine (GemLip) were compared to the conventional (clinical) formulation of gemcitabine (GemConv). METHODS: Gemcitabine was entrapped in a vesicular phospholipid gel (VPG) consisting of densely packed liposomes. Redispersed VPG containing GemLip consisted of 33% liposomally entrapped and 67% free gemcitabine. The in vivo efficacies of GemLip and GemConv were compared using the subcutaneously growing human soft tissue sarcoma SXF 1301 and the orthotopically growing human bladder cancer BXF 1299T. PK and biodistribution were evaluated using radiolabeled drug and lipid in SXF 1301 tumor-bearing nude mice. RESULTS: GemLip was highly active in SXF 1301 at a gemcitabine dose of 6-9 mg/kg (days 1, 8 and 15; dose near the MTD). In the 6-mg/kg groups, complete tumor remissions were observed in seven of eight mice. Equimolar doses of GemConv resulted in only moderate tumor growth inhibition. Even at equitoxic doses (360 mg/kg given on days 1, 8 and 15, or 120 mg/kg on days 1, 5 and 8) GemConv was less active than GemLip. Furthermore, GemLip was active in the orthotopically growing BXF 1299T bladder cancer model at 6 mg/kg and prevented distant organ metastasis. In the PK study, GemLip achieved a 35-fold higher plasma AUC (1680 mg x h/ml) than GemConv (47.6 mg x h/ml). The serum half-lives were 0.15 h for free gemcitabine and 13.3 h for liposomal gemcitabine (6 mg/kg each i.v.). Moreover, gemcitabine levels in tumors were fourfold higher following injection of GemLip than following injection of GemConv. CONCLUSIONS: GemLip is a highly effective gemcitabine delivery system which results in superior gemcitabine pharmacodynamics and PK than GemConv. The enhanced in vivo efficacy might be explained by sustained release and passive tumor targeting.

Animals↗

PubMed2001

Synthetic 2-aroylindole derivatives as a new class of potent tubulin-inhibitory, antimitotic agents.

A new class of simple synthetic antimitotic compounds based on 2-aroylindoles was discovered. (5-Methoxy-1H-2-indolyl)-phenylmethanone (1) as well as analogous 3-fluorophenyl- (36) and 3-methoxyphenyl (3) derivatives displayed high cytotoxicity of IC(50) = 20 to 75 nM against the human HeLa/KB cervical, SK-OV-3 ovarian, and U373 astrocytoma carcinoma cell lines. The inhibition of proliferation correlated with the arrest in the G2/M phase of the cell cycle. In in vitro assays with tubulin isolated from bovine brain, in general antiproliferative activity correlated with inhibition of tubulin polymerization. Thus, the antimitotic activity of 2-aroylindoles is explained by interference with the mitotic spindle apparatus and destabilization of microtubules. In contrast to colchicine, vincristine, nocodazole, or taxol, 1 did not significantly affect the GTPase activity of beta-tubulin. Interestingly, selected compounds inhibited angiogenesis in the chorioallantoic membrane (CAM) assay. In xenograft experiments, 1 was highly active after oral administration at 200 mg/kg against the human amelanocytic melanoma MEXF 989 in athymic nude mice. We conclude, that 2-aroylindoles constitute an interesting new class of antitubulin agents with the potential to be clinically developed for cancer treatment.

Allantois↗

PubMed2001

Genotyping of NAD(P)H:quinone oxidoreductase (NQO1) in a panel of human tumor xenografts: relationship between genotype status, NQO1 activity and the response of xenografts to Mitomycin C chemotherapy in vivo(1).

Pharmacogenetic analysis of polymorphisms in drug metabolizing enzymes is currently generating considerable interest as a means of individualizing patient therapy. Recent studies have suggested that patients that are homozygous for a polymorphic variant (a C to T transition at position 609 of the cDNA sequence) of the enzyme NAD(P)H:quinone oxidoreductase (NQO1) may be resistant to Mitomycin C (MMC). Genotyping of a panel of 54 human tumor xenografts by polymerase chain reaction restriction fragment length polymorphism (PCR-RFLP), classified tumors as wild type (40/54), heterozygotes (11/54), and homozygous mutants (3/54). Previously, 37 of these tumors had been characterized in terms of their response to MMC in vivo, and in this study, a further nine tumor xenografts have been characterized in terms of their response to MMC. No correlation could be found between the NQO1 polymorphic status of xenografts and their response to MMC in vivo. In terms of genotype/phenotype relationships, NQO1 activity in tumors genotyped as wild type, heterozygotes, and homozygous mutants were 311.1 +/- 421.9 (N = 40), 76.9 +/- 109.5 (N = 11), and 0.2 +/- 0.17 (N = 3) nmol/min/mg, respectively. Genotyping of patients may provide a useful initial step in identifying patients who are unlikely to benefit from quinone-based chemotherapy. In the case of MMC, however, the work presented here demonstrates that genotyping of individuals with respect to NQO1 is unlikely to be beneficial in terms of predicting tumor responses to MMC.

Animals↗

PubMed2001

Downregulation of the potential suppressor gene IGFBP-rP1 in human breast cancer is associated with inactivation of the retinoblastoma protein, cyclin E overexpression and increased proliferation in estrogen receptor negative tumors.

The complex insulin-like growth factor network of ligands, receptors and binding proteins has been shown to be disturbed in breast cancer. In addition to defects in proteins controlling cell cycle checkpoints, this type of aberrations could affect tumor growth and survival thereby influencing both tumor aggressiveness and potential response to treatments. We have previously identified the T1A12/mac25 protein, which is identical to the IGFBP-rP1, as a differentially expressed gene product in breast cancer cells compared with normal cells. Here we compare the expression of IGFBP-rP1 in 106 tumor samples with known status of cell cycle aberrations and other clinicopathological data. This was done using a tumor tissue section array system that allows for simultaneous immunohistochemical staining of all samples in parallel. Cytoplasmic staining of variable intensity was observed in most tumors, 15% lacked IGFBP-rP1 staining completely, 20% had weak staining, 32% intermediate and 33% showed strong staining. Low IGFBP-rP1 was associated with high cyclin E protein content, retinoblastoma protein (pRb) inactivation, low bcl-2 protein, poorly differentiated tumors and higher stage. There was a significantly impaired prognosis for patients with low IGFBP-rP1 protein tumors. Interestingly, IGFBP-rP1 showed an inverse association with proliferation (Ki-67%) in estrogen receptor negative tumors as well as in cyclin E high tumors suggesting a separate cell cycle regulatory function for IGFBP-rP1 independent of interaction with the estrogen receptor or the pRb pathway.

Aneuploidy↗

PubMed2001

Pre-clinical evaluation of a methotrexate-albumin conjugate (MTX-HSA) in human tumor xenografts in vivo.

Methotrexate covalently bound to human serum albumin in a 1:1 molar ratio (MTX-HSA) is a new macromolecular drug which is currently being studied in phase I clinical trials by the German Association for Medical Oncology (AIO) Phase I/II study group. Previous studies have shown that MTX-HSA differs favorably from unbound MTX in terms of plasma half-life time, tumor accumulation of albumin and uptake mechanisms into cancer cells. To achieve optimal drug efficacy, repeated treatment cycles were necessary. To evaluate the anti-tumor activity of MTX-HSA and MTX in pre-clinical in vivo models, we selected 7 solid human tumor xenografts growing s.c. in nude mice and administered drug either i.p. or i.v. weekly for 3 weeks. The maximal tolerated dose (MTD) of MTX-HSA in nude mice was 12.5 mg/kg given i.p. on days 1, 8 and 15, whereas the MTD for free MTX was 100 mg/kg given i.v. MTX-HSA was significantly more active (p > 0.01) than MTX in 3 models. In the soft tissue sarcoma SXF 1301, MTX-HSA effected complete remission/cure after a single injection, whereas free MTX resulted in short-lasting, partial tumor regression. In the prostate-cancer model PRXF PC3M, MTX-HSA produced growth inhibition of 92.8% of control or an optimal test/control (T/C) of 7.2% compared to a T/C of 20.8% for MTX (p = 0.05). In the osteosarcoma model SXF 1410, optimal T/C values were 10.2% and 14.5%, respectively (p = 0.025). In lung cancers LXFE 409 and LXFL 529, bladder cancer BXF 1258 and breast cancer MAXF 449, both compounds were inactive. The improved therapeutic effects seen in 3 xenograft models under MTX-HSA treatment are promising and might be due to specific accumulation of the compound in solid tumors owing to their enhanced permeability and retention effect. Thus, clinical development of MTX-HSA will continue and sarcomas as well as prostate cancers will be included as potential target tumors for upcoming clinical phase II trials.

Adult↗

PubMed2001

Differential inhibition of tumor angiogenesis by tie2 and vascular endothelial growth factor receptor-2 dominant-negative receptor mutants.

Tumor growth is angiogenesis-dependent. Current evidence suggests that vascular endothelial growth factor (VEGF), a major regulator of embryonic and hypoxia-mediated angiogenesis, is necessary for tumor angiogenesis. VEGF is expressed in tumor cells in vivo, and its tyrosine kinase receptors VEGFR-1 and VEGFR-2 are up-regulated in the tumor endothelium. A second endothelial cell-specific ligand/receptor tyrosine kinase system, consisting of the tie2 receptor, its activating ligand angiopoietin-1 and the inhibitory ligand angiopoietin-2, has been characterized. We have examined 6 human primary breast-cancer samples and 4 murine breast-cancer cell lines (M6363, M6378, M6444, M6468), transplanted into nude mice, by in situ hybridization and/or Northern analysis. Expression of angiopoietin-1, angiopoietin-2 and tie2 was compared to VEGF and VEGFR-2 expression. Human tumors expressed VEGFR-2 and tie2 but varied considerably in VEGF and angiopoietin-1/-2 expression. In the murine tumor models, we observed high heterogeneity of receptor and ligand expression. M6363 and M6378 tumors were analyzed in detail because they showed different expression of components of the tie2/angiopoietin signaling system. M6363 tumors expressed VEGF, VEGFR-2 and angiopoietin-2 but not tie2 or angiopoietin-1, suggesting activation of VEGFR-2 and inhibition of tie2 signaling pathways, whereas M6378 tumors expressed VEGF, VEGFR-2, tie2 and angiopoietin-1 but little angiopoietin-2, suggesting activation of both VEGFR-2 and tie2 signaling pathways. In vivo studies using truncated dominant-negative tie2 and VEGFR-2 mutants revealed inhibition of M6363 tumor growth by 15% (truncated tie2) and 36% (truncated VEGFR-2), respectively. In contrast, M6378 tumor growth was inhibited by 57% (truncated tie2) and 47% (truncated VEGFR-2), respectively. These findings support the hypothesis that tumor angiogenesis is dependent on VEGFR-2 but suggest that, in addition, tie2-dependent pathways of tumor angiogenesis may exist. For adequate application of angiogenesis inhibitors in tumor patients, analysis of prevailing angiogenesis pathways may be a prerequisite.

Adenocarcinoma, Mucinous↗

PubMed2001

Antiproliferative activity of an aqueous mistletoe extract in human tumor cell lines and xenografts in vitro.

The in vitro antiproliferative activity of an aqueous mistletoe extract (AME) with a defined content of bioactive mistletoe lectin (ML) was tested in 25 human tumor cell lines, including 20 solid and 5 hematological malignancies and 47 human tumor xenografts. The antiproliferative activity of AME was compared to that of the standard cytotoxic agent doxorubicin (CAS 23214-92-8, adriamycin, ADR) using the sulforhodamin B, propidium iodide and soft agar colony forming assays, respectively. AME was highly cytotoxic in solid human tumors with mean IC70 values in the range of 0.17-1 ng ML/ml (2.8-17 pmol bioactive ML). On a molar basis, AME was 3 to 4 logs more potent than ADR and showed differential cytotoxicity towards tumors of the breast, small cell and non-small cell lung, prostate and renal cell cancers. AME was also highly active in hematological malignancies with steep dose response curves resulting in mean IC70 values of 0.12 ng ML/ml (2 pmol). The acute lymphoblastic leukemia cell line HL-60 was the most sensitive, the histiocytic lymphoma cell line U937 the most resistant hematological malignancy. It is important to stress that AME did not induce a biologically relevant increase of cell proliferation in any of the tumor cell lines tested. Our data suggest that AME has in vitro antitumor profiles similar to those of classical anticancer agents. Clear dose-response relationships were found in all of the performed experiments and interesting differential cytotoxicity patterns were observed. Experiments with sensitive tumor types identified in these in vitro studies are currently ongoing in order to demonstrate the anticancer activity of AME in different animal tumor models.

Animals↗

PubMed2000

Predicting tumor responses to mitomycin C on the basis of DT-diaphorase activity or drug metabolism by tumor homogenates: implications for enzyme-directed bioreductive drug development.

Mitomycin C (MMC) is a clinically used anticancer drug that is reduced to cytotoxic metabolites by cellular reductases via a process known as bioreductive drug activation. The identification of key enzymes responsible for drug activation has been investigated extensively with the ultimate aim of tailoring drug administration to patients whose tumors possess the biochemical machinery required for drug activation. In the case of MMC, considerable interest has been centered upon the enzyme DT-diaphorase (DTD) although conflicting reports of good and poor correlations between enzyme activity and response in vitro and in vivo have been published. The principle aim of this study was to provide a definitive answer to the question of whether tumor response to MMC could be predicted on the basis of DTD activity in a large panel of human tumor xenografts. DTD levels were measured in 45 human tumor xenografts that had been characterized previously in terms of their sensitivity to MMC in vitro and in vivo (the in vivo response profile to MMC was taken from work published previously). A poor correlation between DTD activity and antitumor activity in vitro as well as in vivo was obtained. This study also assessed the predictive value of an alternative approach based upon the ability of tumor homogenates to metabolize MMC. This approach is based on the premise that the overall rate of MMC metabolism may provide a better indicator of response than single enzyme measurements. MMC metabolism was evaluated in tumor homogenates (clarified by centrifugation at 1000 x g for 1 min) by measuring the disappearance of the parent compound by HPLC. In responsive [T/C <10% (T/C defined as the relative size of treated and control tumors)] and resistant (T/C >50%) tumors, the mean half life of MMC was 75+/-48.3 and 280+/-129.6 min, respectively. The difference between the two groups was statistically significant (P < 0.005). In conclusion, these results unequivocally demonstrate that response to MMC in vivo cannot be predicted on the basis of DTD activity. Measurement of MMC metabolism by tumor homogenates on the other hand may provide a better indicator of tumor response, and further studies are required to determine whether this approach has real clinical potential in terms of individualizing patient chemotherapy.

Animals↗

PubMed1999

Cellular uptake, cytotoxicity and DNA-binding studies of the novel imidazoacridinone antineoplastic agent C1311.

C1311 is a novel therapeutic agent with potent activity against experimental colorectal cancer that has been selected for entry into clinical trial. The compound has previously been shown to have DNA-binding properties and to inhibit the catalytic activity of topoisomerase II. In this study, cellular uptake and mechanisms by which C1311 interacts with DNA and exerts cytotoxic effects in intact colon carcinoma cells were investigated. The HT29 colon cancer cell line was chosen to follow cellular distribution of C1311 over a time course of 24 h at drug concentrations that just inhibited cell proliferation by 50% or 100%. Nuclear uptake of C1311 and co-localization with lysosomal or mitochondrial dyes was examined by fluorescence microscopy and effects on these cellular compartments were determined by measurement of acid phosphatase levels, rhodamine 123 release or DNA-binding behaviour. The strength and mode of DNA binding was established by thermal melting stabilization, direct titration and viscometric studies of host duplex length. The onset of apoptosis was followed using a TUNEL assay and DNA-fragmentation to determine a causal relationship of cell death. Growth inhibition of HT29 cells by C1311 was concomitant with rapid drug accumulation in nuclei and in this context we showed that the compound binds to duplex DNA by intercalation, with likely A/T sequence-preferential binding. Drug uptake was also seen in lysosomes, leading to lysosomal rupture and a marked increase of acid phosphatase activity 8 h after exposure to C1311 concentrations that effect total growth inhibition. Moreover, at these concentrations lysosomal swelling and breakdown preceded apoptosis, which was not evident up to 24 h after exposure to drug. Thus, the lysosomotropic effect of C1311 appears to be a novel feature of this anticancer agent. As it is unlikely that C1311-induced DNA damage alone would be sufficient for cytotoxic activity, lysosomal rupture may be a critical component for therapeutic efficacy.

Acid Phosphatase↗

PubMed1998

Down-regulation of T1A12/mac25, a novel insulin-like growth factor binding protein related gene, is associated with disease progression in breast carcinomas.

To define genes that are essential to the initiation and progression of breast cancer we utilized subtractive hybridization and differential display cloning techniques and isolated over 950 cDNAs from breast cell-lines derived from matched normal and tumor tissue. Of these, 102 cDNAs were characterized by DNA sequencing and Northern blot analysis. GenBank searches showed that one of these genes, T1A12 is identical to mac25, an insulin-like growth factor-binding protein related gene. Antibodies generated against the C-terminal region of the T1A12/mac25 protein were used to investigate its expression in 60 primary breast tissues. Sections of 12 benign, 16 ductal carcinoma in situ and 32 infiltrating ductal carcinoma specimens were examined. Strong immunoperoxidase staining was observed in luminal epithelial cells of normal lobules and ducts, in apocrine cells of cysts and fibroadenomas. Moderate to weak protein expression was found in hyperplastic and DCIS cells, but no specific staining was detected in invasive carcinoma cells. FISH mapping using a PAC clone localized the T1A12/mac25 gene to 4q12-13. Microsatellite length polymorphism was studied using markers for 4q in paired normal and tumor breast tissues. Thirty-three per cent (10/30) of the samples were found to be polymorphic with D4S189 and D4S231 microsatellite markers and LOH was detected in 50% (5/10) of these informative samples. Our data indicate that T1A12/mac25 expression is abrogated during breast cancer progression concomitant with loss of heterozygosity on chromosome 4q. T1A12/mac25 may therefore have a tumor suppressor-like function and its expression could indicate a disease with a more favorable status, having a better prognosis.

Breast Neoplasms↗

PubMed1998

A human vascular endothelial cell model to study angiogenesis and tumorigenesis.

Endothelial cell biology has recently been the subject of considerable interest in thrombosis and cancer research. However, the successful establishment of immortalized human endothelial cells which retain differentiated cell characteristics has been rare. We have successfully established immortalized human umbilical vein endothelial cells (HUVECs) by human papilloma virus (HPV)-16 E6-E7. HPV-16 E6, E7 and E6-E7 were successfully introduced into HUVEC cells. Both E6 and E7 cultures had an extended lifespan but eventually underwent senescence. E6-E7 cultures 4-5-2G, however, acquired an indefinite lifespan in culture but did not undergo malignant conversion. Telomerase activity was not detected in either E6 or E7 cultures; however, telomerase was detected in E6-E7 4-5-2G cells. The cells exhibited a 'cobblestone' morphology and developed a capillary-like tube structure upon reaching confluence. The 4-5-2G line expressed Factor VIII related antigen and took up DiI-Ac-LDL as markers of endothelial origin. The line expressed integrin subunits (alpha(v)beta3, alph(v)beta5, beta1, alpha2, alpha3, beta4 and alpha6) consistent with an endothelial origin. The higher passage of 4-5-2G line showed a similar intensity of integrin immunostaining to that of primary HUVECS. Subsequent infection of these immortal cells with the Kirsten murine sarcoma virus which contains an activated K-ras oncogene induced morphological transformation that led to the acquisition of invasion capability and neoplastic properties. Telomerase was also detected in the tumorigenic v-Ki-ras transformed cell line. These cell lines should be useful for studies of the molecular mechanisms underlying normal and neoplastic endothelial cell proliferation and migration, and might also provide an in vitro model for development of pharmacologic and gene therapy for cardiovascular thrombosis and cancer.

Cell Line, Transformed↗

PubMed1998

Detection of novel genes that are up-regulated (Di12) or down-regulated (T1A12) with disease progression in breast cancer.

Carcinogenesis is a multistep process and it is believed that seven to ten alterations are needed to convert a normal cell into a maligant one. Thus, identification of these specific genetic lesions and their role in the progression of cancer will lead to more effective methods for early diagnosis, as well as provide targets for treatment and therapeutic intervention. To isolate a number of genes that are differentially expressed in human breast carcinomas we used subtractive hybridization and differential display cloning techniques. By using these methods we obtained 952 clones, characterized 288 clones by restriction mapping, and 105 by Northern blotting and DNA sequencing. Twenty-four clones were found to be previously unidentified, unique genes. Full-length complementary (c)DNA was isolated from clone T1A12 and Di12, and further characterized by computer data search. Polyclonal antibodies were generated against N- and/or C-terminal peptides of the predicted T1A12 and Di12 amino acid sequences and immunohistochemistry was used to delineate gene function in breast cancers. We found that T1A12 is a novel member of the insulin-like growth factor binding protein family and that its expression decreases with disease progression. In 60 primary breast tissues examined, strong T1A12-specific immunoperoxidase staining was observed in luminal epithelial cells of normal lobules or ducts, and in blood vessels. Moderate protein expression was detected in hyperplastic and ductal carcinoma in situ (DCIS) cells, but no staining was found in invasive ductal carcinoma (IDC) cells. On the contrary, by using the same primary breast specimen Di12, a gene which shares no sequence homology with any protein, was found to be overexpressed in IDC cells but was not detectable in normal breast tissues. Specific strong Di12 staining was seen in the cytoplasm, with a slight increase in perinuclear regions of less well differentiated cells similar to those present in ductal carcinomas with poor prognosis. Both the T1A12 and Di12 genes, studied alone or in combination, could prove valuable for understanding the biology of breast cancer, detecting early and late stage disease, and for selecting appropriate treatments.

Breast Neoplasms↗

PubMed1998

Effect of oncogene expression on telomerase activation and telomere length in human endothelial, fibroblast and prostate epithelial cells.

Although strong evidence is mounting that telomerase reactivation and the thereof resulting stabilization of telomeres is a major mechanism for human cells to overcome replicative senescence, a causal relationship linking telomerase activation conclusively to tumorigenesis remains to be established. Thus, the possibility exists that telomerase activation is passively co-selected as tumors develop. To elucidate the function of telomerase during tumorigenesis, we followed telomerase reactivation during immortalization of human primary cell types with in vitro transforming agents and determined the tumorigenic potential of these cells at various stages of transformation. The effects of SV40, v-Ki-ras, HPV-18 and HPV-16 E6/E7 oncoproteins on telomerase expression was examined in primary and immortalized human prostate epithelial (HPE), human prostate fibroblast (HPF), and umbilical vein endothelial cells (HUVEC). All of five SV40-transformed HPE and HPF lines were telomerase positive and had shorter telomeres than primary cells. The two HPV-18 immortalized HPE cell lines also expressed telomerase activity. In contrast, E6 or E7 alone could not produce immortalized HUVEC and did not reactivate telomerase. Life-span, however, was extended. The E6/E7 immortalized HUVEC had telomerase activity and short but stable telomeres. HPE, HPF or HUVEC cells which had been transformed by one oncoprotein alone were not tumorigenic although they had overcome cellular senescence and re-activated telomerase. However, if these cells were transformed by a second agent, either infection with v-Ki-ras or X-ray treatment, they were able to form tumors in nude mice. This suggests that tumorigenesis is a multistep process and that telomerase activation alone is not sufficient for malignant transformation in human cells.

Antigens, Polyomavirus Transforming↗

PubMed1997

Inhibition of telomerase activity by cisplatin in human testicular cancer cells.

Telomerase, a ribonucleoprotein, elongates and/or maintains telomeres by adding TTAGGG tandem repeat sequences using the RNA component of the enzyme as a template. Enzyme activity appears to be associated with cell immortalisation and malignant progression as telomerase activity has been found in the majority of human tumours, but not in most somatic cells or tissues. Telomerase inhibition has, therefore, been proposed as a novel and potentially selective target for therapeutic intervention. Since telomeric tandem repeats as well as the human telomerase RNA component (hTR) and its gene are guanosine-rich, we examined whether the sequence specific, G-Pt-G, cross-linking agent cisplatin is capable of inhibiting telomerase activity. The TRAP assay was used to measure telomerase activity in cisplatin treated cell extracts and RT-PCR strategies used to examine hTR expression after drug exposure. Cisplatin reduced telomerase activity in a specific and concentration-dependent manner in human testicular tumour cells, whilst doxorubicin, bleomycin, methotrexate, melphalan and transplatin had no effect. It is proposed that telomerase inhibition might be a component of the efficacy of cisplatin in the treatment of testicular cancer.

Animals↗