The effect of transcutaneous vagus nerve stimulation on fear generalization and subsequent fear extinction.

Fear overgeneralization is thought to be one of the cardinal processes underlying anxiety disorders, and a determinant of the onset, maintenance and recurrence of these disorders. Animal studies have shown that stimulating the vagus nerve (VNS) affects neuronal pathways implicated in pattern separation and completion, suggesting it may reduce the generalization of a fear memory to novel situations. In a one-day study, 58 healthy students were subjected to a fear conditioning, fear generalization, and fear extinction paradigm. Participants were randomly assigned to receive either transcutaneous auricular VNS (tVNS; final N = 29) or sham stimulation (final N = 29) during the generalization and extinction phases. tVNS did not affect fear generalization, as reflected by US expectancy ratings and fear potentiated startle responses. However, participants who received tVNS reported lower US expectancy ratings to the CS+ during the extinction phase, possibly reflecting a stronger declarative extinction of fear. No effects of tVNS on fear potentiated startle responses during extinction were found. The pattern of findings regarding extinction of declarative fear suggest a facilitating effect of tVNS.

Side-population cells in luminal-type breast cancer have tumour-initiating cell properties, and are regulated by HER2 expression and signalling.

BACKGROUND: The expression of side-population (SP) cells and their relation to tumour-initiating cells (T-ICs) have been insufficiently studied in breast cancer (BC). We therefore evaluated primary cell cultures derived from patients and a panel of human BC cell lines with luminal- or basal-molecular signatures for the presence of SP and BC stem cell markers. METHODS: The SPs from luminal-type BC were analysed for BC T-IC characteristics, including human epidermal growth factor receptor 2 (HER2), ERalpha, IGFBP7 expression and their ability to initiate tumours in non-obese diabetic severe combined immunodeficiency (NOD/SCID) mice. Pharmacological modulators were used to assess the effects of HER2 signalling and breast cancer-resistance protein (BCRP) expression on SPs. RESULTS: The SP was more prevalent in the luminal subtype of BC compared with the basal subtype. HER2 expression was significantly correlated with the occurrence of an SP (r(2)=0.75, P=0.0003). Disappearance of SP in the presence of Ko143, a specific inhibitor of the ATP-binding cassette transporter BCRP, suggests that BCRP is the predominant transporter expressed in this population. The SP also decreased in the presence of HER2 signalling inhibitors AG825 or trastuzumab, strengthening the notion that HER2 contributed to the SP phenotype, likely through downstream AKT signalling. The SP cells from luminal-type MCF-7 cells with enforced expression of HER2, and primary cells with luminal-like properties from a BC patient, displayed enrichment in cells capable of repopulating tumours in NOD/SCID mice. Engraftment of SP cells was inhibited by pretreatment with AG825 or by in vivo treatment with trastuzumab. INTERPRETATION: Our findings indicate an important role of HER2 in regulating SP and hence T-ICs in BC, which may account for the poor responsiveness of HER2-positive BCs to chemotherapy, as well as their aggressiveness.

From ear to eye? No effect of transcutaneous vagus nerve stimulation on human pupil dilation: A report of three studies.

Transcutaneous stimulation of the auricular branch of the vagus nerve (tVNS) has been proposed as a treatment for a spectrum of physical and psychological disorders. One of the proposed working mechanisms of tVNS is a modulatory effect on the locus coeruleus - noradrenaline (LC-NA) network. We tested this hypothesis in humans in a series of three studies: one focusing on high trait worriers, and two in healthy populations. In all three studies, we tested whether tVNS increases resting pupil diameter - as an index of LC-NA network activity. Additionally, we tested whether tVNS affects task performance and task-related pupil dilation during an Attentional Blink task. We found no evidence that tVNS increases pupil diameter or task-related pupil dilation in any of the tasks. No consistent effects of tVNS on performance on the attentional blink task were found. Overall, the results of these studies indicate that tVNS does not affect these behavioral and physiological indices of noradrenergic activity.

Transcutaneous vagus nerve stimulation reduces spontaneous but not induced negative thought intrusions in high worriers.

Worrying is a central component of anxiety disorders. We tested whether non-invasive vagus nerve stimulation reduces negative thought intrusions in high worriers. Worry was assessed with a Breathing Focus Task, which consists of a pre-worry period, a worry induction, and a post-worry period. Ninety-seven high worriers were randomly allocated to receive transcutaneous electrical stimulation of the auricular branch of the vagus nerve at the concha (tVNS), or of the earlobe (sham stimulation) throughout the lab session. Participants who received tVNS reported significantly fewer negative thought intrusions during the pre-worry period, but the effects of tVNS after the worry induction were mixed. An exploratory analysis indicated that participants in the tVNS condition were more likely to report negative thought intrusions shortly after the worry induction, but became less likely to do so as the post-worry period went on. No effects of tVNS on RMSSD were observed. These findings provide preliminary indications that tVNS may decrease the occurrence of worrisome thoughts.

Telomerase and its potential for therapeutic intervention.

Telomerase and telomeres are attractive targets for anticancer therapy. This is supported by the fact that the majority of human cancers express the enzyme telomerase which is essential to maintain their telomere length and thus, to ensure indefinite cell proliferation--a hallmark of cancer. Tumours have relatively shorter telomeres compared to normal cell types, opening the possibility that human cancers may be considerably more susceptible to killing by agents that inhibit telomere replication than normal cells. Advances in the understanding of the regulation of telomerase activity and the telomere structure, as well as the identification of telomerase and telomere associated binding proteins have opened new avenues for therapeutic intervention. Here, we review telomere and telomerase biology and the various approaches which have been developed to inhibit the telomere/telomerase complex over the past decade. They include inhibitors of the enzyme catalytic subunit and RNA component, agents that target telomeres, telomerase vaccines and drugs targeting binding proteins. The emerging role of telomerase in cancer stem cells and the implications for cancer therapy are also discussed.

Preclinical pharmacology of the pyrrolobenzodiazepine (PBD) monomer DRH-417 (NSC 709119).

The pyrrolobenzodiazepine monomer DRH-417 is a member of the anthramycin group of anti-tumor antibiotics that bind covalently to the N2 of guanine within the minor groove of DNA. DRH-417 emerged from the EORTC-Drug Discovery Committee and NCI 60 cell line in vitro screening programs as a potent antiproliferative agent with differential sensitivity towards certain cancer types such as melanoma, breast and renal cell carcinoma (mean IC(50) = 3 nM). DRH-417 was therefore tested for in vivo activity. The maximum tolerated dose (MTD) was established as 0.5 mg/kg given i.p. Marked anti-tumor activity was seen in two human renal cell cancers, one breast cancer and a murine colon tumor model (p<0.01). A selective HPLC (LC/MS) analytical method was developed and plasma pharmacokinetics determined. At a dose of 0.5 mg kg(-1), the plasma AUC was 540 nM h (197.1 ng h ml(-1)) and the peak plasma concentration (171 nM [62.4 ng ml(-1)]) occurred at 30 min., reaching doses levels well above those needed for in vitro antiproliferative activity. Genomic profiling of in vivo sensitive tumors revealed that the latter have an activated insulin-like growth factor signaling pathway.

Mixed evidence for the potential of non-invasive transcutaneous vagal nerve stimulation to improve the extinction and retention of fear.

Extinction memories are fragile and their formation has been proposed to partially rely on vagus nerve activity. We tested whether stimulating the auricular branch of the vagus (transcutaneous VNS; tVNS) accelerates extinction and reduces spontaneous recovery of fear. Forty-two healthy students participated in a 3-day fear conditioning study, where we tested fear acquisition (day 1), fear extinction (day 2) and the retention of the extinction memory (day 3). During extinction, participants were randomly allocated to receive tVNS or sham stimulation concurrently with each CS presentation. During the acquisition and retention phases, all participants received sham stimulation. Indexes of fear included US-expectancy, startle blink EMG and skin conductance responses. Results showed successful acquisition and extinction of fear in all measures. tVNS facilitated the extinction of declarative fear (US expectancy ratings), but did not promote a stronger retention of the declarative extinction memory. No clear effects of tVNS on extinction and retention of extinction were found for the psychophysiological indexes. The present findings provide tentative indications that tVNS could be a promising tool to improve fear extinction and call for larger scale studies to replicate these effects.

Tyrphostin AG17, [(3,5-Di-tert-butyl-4-hydroxybenzylidene)- malononitrile], inhibits cell growth by disrupting mitochondria.

[(3,5-Di-tert-butyl-4-hydroxybenzylidene)-malononitrile] (AG17), a "tyrphostin" tyrosine kinase antagonist, was found to inhibit tumor cell growth with 50% growth inhibition ranging from 0.7 to 4.0 microM in a panel of 13 human tumor cell lines, as evaluated by tetrazolium dye reduction and inhibition of precursor incorporation into macromolecules. The promyelocytic leukemia cell line HL-60(TB), was the most sensitive with irreversible total growth inhibition after 12 h of exposure to 1.5 microM drug. Antiproliferative effects of AG17 in HL-60(TB) cells were temporally related to disruption of mitochondrial function, which occurred within 1 h after drug exposure as demonstrated by a significantly decreased mass of ATP in drug-treated cells, loss of the fluorescent mitochondrial membrane potential probe rhodamine 123, and ultrastructural examination of mitochondria using fluorescence and electron microscopy. Specific decreases of total or tyrosine-phosphorylated substrate at concentrations of the drug not affecting ATP levels were not detected. These data raise the possibility that AG17 may act in part by altering mitochondrial function and/or structure, and that impairment of mitochondrial function may be exploitable as a potentially useful mechanism to modulate tumor cell proliferation. This study also emphasizes the importance of evaluating carefully the effects of potential protein kinase antagonists, since these structures have effects in intact cells in addition to what might be expected from in vitro enzyme assays.

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.

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.

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.

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.

Antiproliferative activity in vitro and in vivo of the spicamycin analogue KRN5500 with altered glycoprotein expression in vitro.

The spicamycin analogue KRN5500 (NSC 650426; SPA) is derived from Streptomyces alanosinicus. The unique structure contains a purine, an aminoheptose sugar, glycine, and a tetradecadiene fatty acid. SPA potently inhibits the growth of certain human tumor cell lines in vitro (IC50 for growth <100 nM) and displays marked activity in vivo in Colo 205 colon carcinoma xenografts. Selective inhibition of labeled precursor incorporation was not evident at 1 or 4 h of exposure to the drug, but at 8 h, [3H] leucine incorporation was inhibited by approximately 40% at or below the IC50 for cell growth. Because of the structural similarity of SPA to inhibitors of glycoprotein processing, we examined the effect of SPA on indicators of glycoprotein synthesis and processing in HL60TB promyelocytic leukemia and Colo 205 colon carcinoma cells. Brief periods of exposure ( approximately 30 min) to SPA at the IC50 for growth increased incorporation of [3H]mannose. When examined by Western blotting after prolonged (40-48 h) incubation with lectins that target mannose-containing carbohydrates, Galanthus nivalis agglutinin and concanavalin A, a qualitative change in the pattern of mannose-containing glycoproteins was observed in HL60TB cells. Significant changes in the pattern of surface glycoprotein expression in intact cells were demonstrated by flow cytometry using fluorescence-labeled lectins. An increase in the number of cells binding G. nivalis agglutinin (indicating terminal mannose) was noted, but a decrease in the amount of lectin bound per cell was noted for wheat germ agglutinin (detecting sialic acid and terminal beta-N-acetyl glucosamine residues). Electron microscopy revealed loss of microvilli, and the Golgi apparatus appeared inflated. Our findings, therefore, raise the possibility that cells exposed to SPA have altered glycoprotein processing after exposure to low concentrations of drug, prior to the occurrence of overt cytotoxicity. These effects are consistent with a prominent early effect of SPA on the enzymatic machinery or organelles important for proper glycoprotein processing and emphasize the novelty of this agent's likely mechanism of action.

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.

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.

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.

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.

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.

Telomerase in cancer diagnosis and therapy: a clinical perspective.

Curing cancers is one of the most challenging tasks of modern medicine. The major problem is the heterogeneity of human tumours and thus finding a 'universal' target for cancer treatment. The discovery that the expression of the enzyme telomerase is a hallmark of immortality and cancer, and that it is found in the majority (>85%) of human tumours but is repressed in most normal cells, has therefore caused considerable excitement. These observations led to the design of potential telomerase inhibitors and ideas about targeting telomerase in the clinic. To date, several classes of telomerase inhibitory agents have been identified and are in preclinical development. However, the approach has not yet been tested clinically. Because of the proposed function of telomerase, and the understanding that replicative cell senescence or cell death result from progressive telomere shortening during successive cell divisions, even complete enzyme inhibition will not produce immediate cell death. Designing clinical trials for promising telomerase inhibitors requires consideration of the novel mechanism of action of these drugs. A lag period between initiation of treatment and occurrence of effects is likely, and thus anti-telomerase therapy might best be given in adjuvant treatment protocols after initial tumour debulking therapy and in combination with other cytostatic agents. The available knowledge of telomerase biology and its association with human tumours suggests that telomerase inhibition might prove a valuable addition to current cancer treatment regimens.

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.