Melphalan, prednisone, and lenalidomide versus melphalan, prednisone, and thalidomide in untreated multiple myeloma.

The combination of melphalan, prednisone, and thalidomide (MPT) is considered standard therapy for newly diagnosed patients with multiple myeloma who are ineligible for stem cell transplantation. Long-term treatment with thalidomide is hampered by neurotoxicity. Melphalan, prednisone, and lenalidomide, followed by lenalidomide maintenance therapy, showed promising results without severe neuropathy emerging. We randomly assigned 668 patients between nine 4-week cycles of MPT followed by thalidomide maintenance until disease progression or unacceptable toxicity (MPT-T) and the same MP regimen with thalidomide being replaced by lenalidomide (MPR-R). This multicenter, open-label, randomized phase 3 trial was undertaken by Dutch-Belgium Cooperative Trial Group for Hematology Oncology and the Nordic Myeloma Study Group (the HOVON87/NMSG18 trial). The primary end point was progression-free survival (PFS). A total of 318 patients were randomly assigned to receive MPT-T, and 319 received MPR-R. After a median follow-up of 36 months, PFS with MPT-T was 20 months (95% confidence interval [CI], 18-23 months) vs 23 months (95% CI, 19-27 months) with MPR-R (hazard ratio, 0.87; 95% CI, 0.72-1.04; P = .12). Response rates were similar, with at least a very good partial response of 47% and 45%, respectively. Hematologic toxicity was more pronounced with MPR-R, especially grades 3 and 4 neutropenia: 64% vs 27%. Neuropathy of at least grade 3 was significantly higher in the MPT-T arm: 16% vs 2% in MPR-R, resulting in a significant shorter duration of maintenance therapy (5 vs 17 months in MPR-R), irrespective of age. MPR-R has no advantage over MPT-T concerning efficacy. The toxicity profile differed with clinically significant neuropathy during thalidomide maintenance vs myelosuppression with MPR.

Autologous/reduced-intensity allogeneic stem cell transplantation vs autologous transplantation in multiple myeloma: long-term results of the EBMT-NMAM2000 study.

Long-term follow-up of prospective studies comparing allogeneic transplantation to autologous transplantation in multiple myeloma is few and controversial. This is an update at a median follow-up of 96 months of the European Group for Blood and Marrow Transplantation Non-Myeloablative Allogeneic stem cell transplantation in Multiple Myeloma (NMAM)2000 study that prospectively compares tandem autologous/reduced intensity conditioning allogeneic transplantation (auto/RICallo) to autologous transplantation alone (auto). There are 357 myeloma patients up to age 69 years enrolled. Patients with an HLA-identical sibling were allocated to auto/RICallo (n = 108) and those without to auto alone (n = 249). At 96 months progression-free survival (PFS) and overall survival (OS) were 22% and 49% vs 12% (P = .027) and 36% (P = .030) with auto/RICallo and auto respectively. The corresponding relapse/progression rate (RL) was 60% vs 82% (P = .0002). Non-relapse mortality at 36 months was 13% vs 3% (P = .0004). In patients with the del(13) abnormality corresponding PFS and OS were 21% and 47% vs 5% (P = .026), and 31% (P = .154). Long-term outcome in patients with multiple myeloma was better with auto/RICallo as compared with auto only and the auto/RICallo approach seemed to overcome the poor prognostic impact of del(13) observed after autologous transplantation. Follow up longer than 5 years is necessary for correct interpretation of the value of auto/RICallo in multiple myeloma.

Bortezomib consolidation after autologous stem cell transplantation in multiple myeloma: a Nordic Myeloma Study Group randomized phase 3 trial.

The Nordic Myeloma Study Group conducted an open randomized trial to compare bortezomib as consolidation therapy given after high-dose therapy and autologous stem cell transplantation (ASCT) with no consolidation in bortezomib-naive patients with newly diagnosed multiple myeloma. Overall, 370 patients were centrally randomly assigned 3 months after ASCT to receive 20 doses of bortezomib given during 21 weeks or no consolidation. The hypothesis was that consolidation therapy would prolong progression-free survival (PFS). The PFS after randomization was 27 months for the bortezomib group compared with 20 months for the control group (P = .05). Fifty-one of 90 patients in the treatment group compared with 32 of 90 controls improved their response after randomization (P = .007). No difference in overall survival was seen. Fatigue was reported more commonly by the bortezomib-treated patients in self-reported quality-of-life (QOL) questionnaires, whereas no other major differences in QOL were recorded between the groups. Consolidation therapy seemed to be beneficial for patients not achieving at least a very good partial response (VGPR) but not for patients in the ≥ VGPR category at randomization. Consolidation with bortezomib after ASCT in bortezomib-naive patients improves PFS without interfering with QOL. This trial was registered at www.clinicaltrials.gov as #NCT00417911.

Improved survival in myeloma patients: starting to close in on the gap between elderly patients and a matched normal population.

The outcome for multiple myeloma patients has improved since the introduction of bortezomib, thalidomide and lenalidomide. However, studies comparing new and conventional treatment include selected patient groups. We investigated consecutive patients (n = 1638) diagnosed in a defined period and compared survival with a gender- and age-matched cohort Swedish population (n = 9 340 682). Median overall survival for non-high-dose treated patients was 2·8 years. The use of bortezomib, thalidomide or lenalidomide in first line therapy predicted a significantly longer overall survival (median 4·9 years) compared to conventional treatment (2·3 years). Among non-high-dose treated patients receiving at least 2 lines with bortezomib, thalidomide or lenalidomide, 69% and 63% have survived at 3 and 5 years as compared to 48% and 22% with conventional drugs and 88% and 79% in the matched cohort populations, respectively. The median overall survival in high-dose treated patients was 6·9 years. Of these patients, 84% survived at 3 years and 70% at 5 years as compared to 98% and 95% in the matched cohort population. Overall survival in the best non-high-dose treated outcome group is closing the gap with the matched cohort. Upfront use of new drugs is clearly better than waiting until later lines of treatment.

The N-terminal region of comparative gene identification-58 (CGI-58) is important for lipid droplet binding and activation of adipose triglyceride lipase.

In mammals, excess energy is stored in the form of triacylglycerol primarily in lipid droplets of white adipose tissue. The first step of lipolysis (i.e. the mobilization of fat stores) is catalyzed by adipose triglyceride lipase (ATGL). The enzymatic activity of ATGL is strongly enhanced by CGI-58 (comparative gene identification-58), and the loss of either ATGL or CGI-58 function causes systemic triglyceride accumulation in humans and mice. However, the mechanism by which CGI-58 stimulates ATGL activity is unknown. To gain insight into CGI-58 function using structural features of the protein, we generated a three-dimensional homology model based on sequence similarity with other proteins. Interestingly, the model of CGI-58 revealed that the N terminus forms an extension of the otherwise compact structure of the protein. This N-terminal region (amino acids 1-30) harbors a lipophilic tryptophan-rich stretch, which affects the localization of the protein. (1)H NMR experiments revealed strong interaction between the N-terminal peptide and dodecylphosphocholine micelles as a lipid droplet-mimicking system. A role for this N-terminal region of CGI-58 in lipid droplet binding was further strengthened by localization studies in cultured cells. Although wild-type CGI-58 localizes to the lipid droplet, the N-terminally truncated fragments of CGI-58 are dispersed in the cytoplasm. Moreover, CGI-58 lacking the N-terminal extension loses the ability to stimulate ATGL, implying that the ability of CGI-58 to activate ATGL is linked to correct localization. In summary, our study shows that the N-terminal, Trp-rich region of CGI-58 is essential for correct localization and ATGL-activating function of CGI-58.

Chromatin remodeling: recruitment of histone demethylase RBP2 by Mad1 for transcriptional repression of a Myc target gene, telomerase reverse transcriptase.

The Myc/Max/Mad network transcription factors are known to govern target gene expression through recruiting histone acetyltransferases or deacetylases. In the present study, we show that Mad1 recruits the histone demethylase RBP2 to the Myc target telomerase reverse transcriptase (hTERT) gene promoter to repress transcription. With differentiation of leukemic HL60 cells, Mad1 and RBP2 were both up-regulated, interacted, and cooccupied the hTERT promoter accompanied by histone H3-K4 demethylation. In immortalized p493-6 B cells, shutting down c-Myc led to the accumulation of Mad1 and RBP2 at hTERT promoter and diminished hTERT mRNA expression. When RBP2 was depleted, hTERT expression was significantly enhanced, coupled with dissociation of RBP2 with and increased H3-K4 methylation at the hTERT promoter in p493-6 cells. Moreover, RBP2 and Mad1 were present on the hTERT promoter in human fibroblasts having a silent hTERT gene, and RBP2 depletion resulted in gene derepression. Taken together, Mad1 recruits RBP2 to the hTERT promoter that, in turn, demethylates H3-K4, thereby contributing to a stable repression of the hTERT gene in normal or differentiated malignant cells. Our findings reveal a novel mechanism through which the Myc/Max/Mad network proteins control their target gene transcription and provide insights into mechanisms underlying telomerase silencing and activation.

Inverse relationship between leukaemic cell burden and plasma concentrations of daunorubicin in patients with acute myeloid leukaemia.

AIMS: It has been shown that the cellular uptake and cytotoxicity of anthracyclines decrease with increasing cell density in vitro, an event termed 'the inocculum effect'. It is not known whether such an effect occurs in vivo. In this study the relationships between white blood cell (WBC) count, plasma and cellular concentrations of daunorubicin (DNR) in patients with acute myeloid leukaemia were investigated. METHODS: Plasma and mononuclear blood cells were isolated from peripheral blood from 40 patients with acute myeloid leukaemia at end of infusion (time 1 h), 5 and 24 h following the first DNR infusion. DNR concentrations were determined by high-pressure liquid chromatography and related to the WBC count at diagnosis. A population pharmacokinetic model was used to estimate the correlations between baseline WBC count, volume of distribution and clearance of DNR. RESULTS: A clear but weak inverse relationship between the baseline WBC count and plasma concentrations of DNR (r(2)=0.11, P<0.05) at time 1 was found. Furthermore, a clear relationship between baseline WBC count and DNR central volume of distribution using population pharmacokinetic modelling (dOFV 4.77, P<0.05) was also noted. Analysis of plasma DNR and the metabolite daunorubicinol (DOL) concentrations in patients with a high WBC count support that the low DNR/DOL concentrations are due a distribution effect. CONCLUSION: This study shows that the leukaemic cell burden influences the plasma concentrations of anthracyclines. Further studies are needed to explore if patients with high a WBC count may require higher doses of anthracyclines.

Mitogen-activated protein kinase cascade-mediated histone H3 phosphorylation is critical for telomerase reverse transcriptase expression/telomerase activation induced by proliferation.

Telomerase activity and telomerase reverse transcriptase (hTERT), the key component of the telomerase complex, are tightly proliferation regulated in normal and malignant cells both in vitro and in vivo; however, underlying mechanisms are unclear. In the present study, we identified mitogen-activated protein kinase (MAPK) cascade-mediated histone H3 ser10 phosphorylation to be a molecular link between proliferation and induction of hTERT/telomerase activity. In normal human T lymphocytes and fibroblasts, growth or stress stimuli known to drive H3 phosphorylation through the MAPK signaling induce hTERT expression and/or telomerase activity that was preceded by phosphorylated histone H3 (ser10) at the hTERT promoter. Blockade of the MAPK-triggered H3 phosphorylation significantly abrogates hTERT induction and ser10 phosphorylation at this promoter. However, H3 ser10 phosphorylation alone resulted in low, transient hTERT induction, as seen in fibroblasts, whereas H3 phosphorylation followed by its acetylation at lys14 robustly trans-activated the hTERT gene accompanying constitutive telomerase activity in normal and malignant T cells. H3 acetylation without phosphorylation similarly exerted weak effects on hTERT expression. These results define H3 phosphorylation as a key to hTERT transactivation induced by proliferation and reveal a fundamental mechanism for telomerase regulation in both normal human cells and transformed T cells.

Uptake of anthracyclines in vitro and in vivo in acute myeloid leukemia cells in relation to apoptosis and clinical response.

AIMS: To study anthracycline-induced apoptosis in leukemic cells isolated from patients with acute myelogenous leukemia (AML) in vitro and to compare intracellular anthracycline concentrations causing apoptosis in vitro with those obtained in vivo during anthracycline treatment. METHODS: Mononuclear blood cells from AML patients were isolated before (n = 20) and after anthracycline infusion (n = 24). The pre-treated cells were incubated in vitro with daunorubicin (DNR) and/or idarubicin (IDA). Anthracycline concentrations were determined by high-performance liquid chromatography, and apoptosis was detected by propidium iodine staining using a flow cytometer. RESULTS: There was a clear concentration-response relationship between intracellular anthracycline levels and apoptosis albeit with a large interindividual variation. Intracellular levels >1200 muM always led to high apoptosis development (>60%) in vitro. The intracellular concentrations of DNR in vivo (n = 24) were more than tenfold lower than the concentrations needed to induce effective apoptosis in vitro, although a significant relation between in vivo concentrations and clinical remission was found. We also found a significant relation between apoptosis induction in leukemic cells by IDA in vitro and clinical remission. CONCLUSIONS: Our results indicate that intracellular anthracycline levels in vivo are suboptimal and that protocols should be used that increase intracellular anthracycline levels.

The telomerase reverse transcriptase (hTERT) gene is a direct target of the histone methyltransferase SMYD3.

Recent evidence has accumulated that the dynamic histone methylation mediated by histone methyltransferases and demethylases plays key roles in regulation of chromatin structure and transcription. In the present study, we show that SET and MYND domain-containing protein 3 (SMYD3), a histone methyltransferase implicated in oncogenesis, directly trans-activates the telomerase reverse transcriptase (hTERT) gene that is essential for cellular immortalization and transformation. SMYD3 occupies its binding motifs on the hTERT promoter and is required for maintenance of histone H3-K4 trimethylation, thereby contributing to inducible and constitutive hTERT expression in normal and malignant human cells. Knocking down SMYD3 in tumor cells abolished trimethylation of H3-K4, attenuated the occupancy by the trans-activators c-MYC and Sp1, and led to diminished histone H3 acetylation in the hTERT promoter region, which was coupled with down-regulation of hTERT mRNA and telomerase activity. These results suggest that SMYD3-mediated trimethylation of H3-K4 functions as a licensing element for subsequent transcription factor binding to the hTERT promoter. The present findings provide significant insights into regulatory mechanisms of hTERT/telomerase expression; moreover, identification of the hTERT gene as a direct target of SMYD3 contributes to a better understanding of SMYD3-mediated cellular transformation.

Infectious complications and NK cell depletion following daratumumab treatment of Multiple Myeloma.

Treatment with Daratumumab (Dara), a monoclonal anti-CD38 antibody of IgG1 subtype, is effective in patients with multiple myeloma (MM). However, Dara also impairs the cellular immunity, which in turn may lead to higher susceptibility to infections. The exact link between immune impairment and infectious complications is unclear. In this study, we report that nine out of 23 patients (39%) with progressive MM had infectious complications after Dara treatment. Five of these patients had viral infections, two developed with bacterial infections and two with both bacterial and viral infections. Two of the viral infections were exogenous, i.e. acute respiratory syncytial virus (RSV) and human metapneumovirus (hMPV), while five consisted of reactivations, i.e. one herpes simplex (HSV), 1 varicella-zoster (VZV) and three cytomegalovirus (CMV). Infections were solely seen in patients with partial response or worse. Assessment of circulating lymphocytes indicated a selective depletion of NK cells and viral reactivation after Dara treatment, however this finding does not exclude the multiple components of viral immune-surveillance that may get disabled during this monoclonal treatment in this patient cohort. These results suggest that the use of antiviral and antibacterial prophylaxis and screening of the patients should be considered.

All-oral ixazomib, cyclophosphamide, and dexamethasone for transplant-ineligible patients with newly diagnosed multiple myeloma.

BACKGROUND: Novel efficacious treatments with long-term tolerability are needed for transplant-ineligible, newly diagnosed multiple myeloma (NDMM) patients. This phase 2 study evaluated the safety and efficacy of all-oral ixazomib-cyclophosphamide-dexamethasone (ICd) followed by single-agent ixazomib maintenance. PATIENTS AND METHODS: Patients were randomised (1:1) to receive 4.0 mg of ixazomib, 300 (Arm A) or 400 (Arm B) mg/m2 of cyclophosphamide (days 1, 8, and 15), and 40 mg of dexamethasone (days 1, 8, 15, and 22) as induction (up to 13 × 28-day cycles), followed by single-agent ixazomib maintenance (28-day cycles) until progressive disease, death, or unacceptable toxicity. Primary end-point was complete response (CR) + very good partial response (VGPR) rate for ICd induction. RESULTS: Seventy patients were enrolled (n = 36 Arm A; n = 34 Arm B); median age was 73 years (range, 61-87). At data cut-off, 66% of patients had completed 13 induction cycles followed by ixazomib maintenance. Median overall treatment duration was 19 cycles (range, 1-29); 21% of patients discontinued treatment during induction and 3% during maintenance due to adverse events (AEs). During induction, among 67 response-evaluable patients, CR+VGPR rate was 25%, and overall response rate (ORR) was 73%. Including the maintenance phase, CR+VGPR rate was 33%, and ORR was 76%. Median progression-free survival was 23.5 months (median follow-up: 26.1 months). The most common all-grade AE was neutropenia (31%). Grade ≥3 AEs were reported by 73% of patients. Five on-study deaths occurred (not treatment-related). CONCLUSIONS: ICd treatment followed by ixazomib maintenance is tolerable and active in elderly, transplant-ineligible NDMM patients. TRIAL REGISTRATION NUMBER: NCT02046070.

Pharmacogenetic study of the impact of ABCB1 single-nucleotide polymorphisms on lenalidomide treatment outcomes in patients with multiple myeloma: results from a phase IV observational study and subsequent phase II clinical trial.

PURPOSE: Despite therapeutic advances, patients with multiple myeloma (MM) continue to experience disease relapse and treatment resistance. The gene ABCB1 encodes the drug transporter P-glycoprotein, which confers resistance through drug extrusion across the cell membrane. Lenalidomide (Len) is excreted mainly via the kidneys, and, given the expression of P-gp in the renal tubuli, single-nucleotide polymorphisms (SNPs) in the ABCB1 gene may influence Len plasma concentrations and, subsequently, the outcome of treatment. We, therefore, investigated the influence of ABCB1 genetic variants on Len treatment outcomes and adverse events (AEs). METHODS: Ninety patients with relapsed or refractory MM, who received the second-line Len plus dexamethasone in the Rev II trial, were genotyped for the ABCB1 SNPs 1199G>A (Ser400Asn, rs2229109), 1236C>T (silent, rs1128503), 2677G>T/A (Ala893Ser, rs2032582), and 3435C>T (silent, rs1045642) using pyrosequencing, and correlations to response parameters, outcomes, and AEs were investigated. RESULTS: No significant associations were found between genotype and either best response rates or hematological AEs, and 1236C>T, 2677G>T or 3435C>T genotypes had no impact on survival. There was a trend towards increased time to progression (TTP) in patients carrying the 1199A variant, and a significant difference in TTP between genotypes in patients with standard-risk cytogenetics. CONCLUSIONS: Our findings show a limited influence of ABCB1 genotype on lenalidomide treatment efficacy and safety. The results suggest that 1199G>A may be a marker of TTP following Len treatment in standard-risk patients; however, larger studies are needed to validate and clarify the relationship.

Lenalidomide versus lenalidomide + dexamethasone prolonged treatment after second-line lenalidomide + dexamethasone induction in multiple myeloma.

Lenalidomide (Len) plus dexamethasone (Dex) is approved for the treatment of relapsed or refractory multiple myeloma (RRMM). It is possible that single-agent Len may be effective as prolonged treatment regimen in RRMM once patients demonstrate an initial response to Len+Dex induction. Patients with RRMM who responded to first-line Len+Dex in an observational study (NCT01430546) received up to 24 cycles of either Len (25 mg/day) or Len+Dex (25 mg/day and 40 mg/week) as prolonged treatment in a subsequent phase 2 clinical trial (NCT01450215). In the observational study (N = 133), median time to response was 1.7 (range 0.6-9.6) months. A complete response to all treatments received in both studies was observed in 11% of patients; very good partial response and partial response rates were 31% and 38%, respectively. Corresponding response rates in the subgroup of patients who did not enter the phase 2 trial (n = 71) were 3%, 18%, and 39%, respectively. Rates of disease progression at 2 years in the phase 2 trial were 47% versus 31% for Len versus Len+Dex (P = 0.14). After 36 months median follow-up in surviving patients, median time to progression was not reached with Len+Dex and was 24.9 months (95% confidence interval 12.5-not calculable, P < 0.001) with Len. Three-year OS among the total observational study population was 61% (95% CI, 52-69%). The corresponding rate among patients who entered the phase 2 clinical trial was 73% (95% CI, 60-83%) and was significantly lower among those patients who achieved ≥PR but did not proceed into the phase 2 trial (55%; P = 0.01). In the phase 2 trial, OS was 73% in both treatment arms (P = 0.70). Neutropenia and thrombocytopenia were more common with prolonged (phase 2 trial) versus short-term (observational study) Len administration but remained manageable. Prolonged treatment with Len with or without Dex provides sustained, clinically relevant responses and demonstrates an acceptable safety profile.

Acute Myelogenous Leukemia Cells Secrete Factors that Stimulate Cellular LDL Uptake via Autocrine and Paracrine Mechanisms.

Leukemic cells isolated from most patients with acute myelogenous leukemia (AML) have higher low density lipoprotein (LDL) uptake than normal mononuclear blood cells. Little is known, however, about the mechanism behind the elevated LDL uptake. We investigated if AML cells secrete factors that stimulate cellular LDL uptake. Mononuclear blood cells were isolated from peripheral blood from 42 patients with AML at diagnosis. Cellular LDL uptake was determined from the degradation rate of 125I-labelled LDL. Conditioned media from AML cells stimulated the LDL degradation in the leukemic cell lines KG1 and HL60, and in isolated AML cells. The stimulatory effect correlated with the LDL degradation in the AML cells directly after isolation from blood. Conditioned media also autostimulated LDL degradation in the AML cells themselves. Concentrations of IL-6 and IL-8 in AML cell conditioned media correlated with the LDL degradation in AML cells directly after isolation from blood. Addition of R-TNF-α, but not IL-6 or IL-8, stimulated LDL degradation in HL60, KG1, and AML cells. The LDL degradation in AML cells could be inhibited by a LDL receptor blocking antibody. AML cells secrete factors that stimulate LDL uptake in a paracrine and autocrine pattern which open up therapeutic possibilities to inhibit the uptake of LDL by administration of antibodies to these factors.

Differential expression of full-length telomerase reverse transcriptase mRNA and telomerase activity between normal and malignant renal tissues.

Activation of telomerase, a key event during immortalization and malignant transformation, requires expression of the telomerase reverse transcriptase (hTERT). Consistently, lack of telomerase activity and hTERT expression occurs in most normal human somatic cells. However, it has been observed that both normal and cancerous renal tissues express hTERT whereas only the latter exhibits telomerase activity. The mechanism underlying the dissociation between hTERT expression and telomerase activity is unclear. In the present study, we examined telomerase activity and alternative splicing of hTERT transcripts in renal cell carcinoma (RCC) specimens and adjacent normal tissues from 33 patients with RCC. Telomerase activity was detectable in 27 of 33 (82%) RCC samples but none in their normal counterparts. Thirty-two of 33 tumors expressed overall hTERT mRNA and 27 of them contained full-length hTERT transcripts, all with telomerase activity. Although 42% (14 of 33) of normal renal samples expressed hTERT mRNA, none of them had full-length hTERT transcripts, coinciding with lack of telomerase activity. The presence of full-length hTERT mRNA and telomerase activity was significantly associated with c-MYC induction. In tumors, absence of full-length hTERT mRNA or telomerase activity defines a subgroup of nonmetastatic, early-stage RCCs. Taken together, telomerase repression in normal renal tissues is attributed to the absence of full-length hTERT transcripts, whereas telomerase activation is achieved via induction of or switch to expression of full-length hTERT mRNA during the oncogenic process of kidneys, and associated with aggressive RCCs.

Telomere attrition predominantly occurs in precursor lesions during in vivo carcinogenic process of the uterine cervix.

Although human papillomavirus (HPV) has been defined as the pathogen for cervical carcinomas, molecular events underlying the oncogenic process are unclear. As telomere dysfunction-mediated chromosomal instability and telomerase activation have been suggested as key events in carcinogenesis, we dissected the dynamic changes in telomere length, checkpoint response, and temporal profile of telomerase expression during the evolution from precursor lesions (cervical intraepithelial neoplasia, CINs) to invasive cancers of the uterine cervix in sequential samples from 16 patients. Telomeres were significantly shortened in all CIN samples and no further substantial attritions occurred in most cases with the acquisition of malignant phenotype. Very short telomeres were coupled with constitutive activation of the DNA damage response pathway (Chk2 phosphorylation) and increased cellular proliferation in those cervical specimens. Telomerase reverse transcriptase (hTERT) expression was preferably induced at advanced CINs or invasive cancers. The present finding demonstrates that excessive telomere shortening predominantly occurs in the early carcinogenesis of the uterine cervix largely prior to telomerase activation. Widespread over-erosion of telomeres or telomere dysfunction in very early stages of cervical tumorigenesis might fuel transformation processes by driving chromosomal instability.

Relationship between daunorubicin concentration and apoptosis induction in leukemic cells.

Aiming to determine if a concentration window exists in which apoptosis induction by daunorubicin (DNR) is optimal, we studied the relationship between DNR concentration and apoptosis induction in HL60 and K562 cells and in peripheral leukemic cells isolated from three patients with acute myelogenous leukemia (AML). Cells were incubated for 2hr with increasing DNR concentrations and thereafter for 22hr in drug-free medium. Apoptosis was measured by detection of caspase-3-like activity and DNA fragmentation assayed by propidium iodide and flow cytometry. High DNR concentrations initiated faster apoptosis in HL60 cells and in AML cells, as shown by caspase-3 and DNA fragmentation data. DNA fragmentation into small fragments was preceded by the formation of a narrow peak on the left side of the G1 peak, most likely large DNA fragments, but further studies are required for unequivocal confirmation. This peak could easily be misinterpreted as a G1 peak without careful time monitoring. In K562 cells, no left peak was detected, apoptosis was slow and not related to concentration. In AML cells, large interindividual variations were observed in the time course of DNA fragmentation at 0.25microg DNR/mL. In conclusion, our findings support the concept of dose intensification for optimal apoptosis induction as higher doses correlate with earlier and more rapid caspase-3 induction and DNA fragmentation in leukemic cells. The DNA fragmentation assay may be a valuable tool to determine leukemic cells' chemosensitivity to apoptosis.

Different effects of metabolic inhibitors and cyclosporin A on daunorubicin transport in leukemia cells from patients with AML.

The objective of this study was to determine the role of transport proteins in daunorubicin (Dnr) accumulation and efflux in leukemia cells from 36 patients with acute myeloid leukemia (AML). Mononuclear cells were isolated and incubated with 1 microM Dnr with/without addition of 3 microM cyclosporin A (CyA) or metabolic inhibitors (MI). Cellular Dnr concentration in leukemia blast cells was measured with flow cytometry. After washing and reincubation of the cells in drug-free medium, Dnr efflux was followed with/without addition of CyA or MI. Levels of mRNA expression for mdr1, multidrug resistance associated protein (mrp) and lung resistance protein (lrp) were determined with reverse transcriptase-polymerase chain reaction (RT-PCR). MI enhanced cellular Dnr accumulation to a higher extent than CyA whereas CyA reduced Dnr efflux more efficiently than MI (P<0.001). There was a significant difference in Dnr accumulation between samples with low and high mdr1 mRNA levels but only in the presence of MI or CyA. Our results imply that other factors than P-glycoprotein (Pgp) are of major importance for in vitro Dnr accumulation in AML blasts and that the role of Pgp as a drug efflux pump is not conclusive.

Real-time RT-PCR for the determination of topoisomerase II mRNA level in leukaemic cells.

We developed a real-time RT-PCR assay for the quantification of topoisomerase II (topo II) mRNA level. It was applied on peripheral leukaemic cells from 23 patients with acute myelogenous leukaemia (AML) and 23 with chronic lymphocytic leukaemia (CLL). RNA template dilutions from 0.25 to 25ng per reaction were used as standard curves for topo IIalpha, beta and the internal control 18S rRNA. About 57% (26/46) and 26% (12/46) of the specimens had detectable topo IIbeta and alpha mRNA, respectively. The correlation between these two factors was rho=0.7 and P=0.0001. No relationship between topo IIalpha or beta mRNA level and response to chemotherapy was found in AML patients (n=19 assessable for response). Our method is rapid and convenient for quantification of topo IIalpha and beta mRNA levels, and could be suitable for investigation in a larger population.