Metastatic renal cell carcinomas (mRCC) are highly vascularized tumors that are a paradigm for the treatment with antiangiogenesis drugs targeting the vascular endothelial growth factor (VEGF) pathway. The available drugs increase the time to progression but are not curative and the patients eventually relapse. In this study we have focused our attention on the molecular mechanisms leading to resistance to sunitinib, the first line treatment of mRCC. Because of the anarchic vascularization of tumors the core of mRCC tumors receives only suboptimal concentrations of the drug. To mimic this in vivo situation, which is encountered in a neoadjuvant setting, we exposed sunitinib-sensitive mRCC cells to concentrations of sunitinib below the concentration of the drug that gives 50% inhibition of cell proliferation (IC50). At these concentrations, sunitinib accumulated in lysosomes, which downregulated the activity of the lysosomal protease CTSB (cathepsin B) and led to incomplete autophagic flux. Amino acid deprivation initiates autophagy enhanced sunitinib resistance through the amplification of autolysosome formation. Sunitinib stimulated the expression of ABCB1 (ATP-binding cassette, sub-family B [MDR/TAP], member 1), which participates in the accumulation of the drug in autolysosomes and favor its cellular efflux. Inhibition of this transporter by elacridar or the permeabilization of lysosome membranes with Leu-Leu-O-methyl (LLOM) resensitized mRCC cells that were resistant to concentrations of sunitinib superior to the IC50. Proteasome inhibitors also induced the death of resistant cells suggesting that the ubiquitin-proteasome system compensates inhibition of autophagy to maintain a cellular homeostasis. Based on our results we propose a new therapeutic approach combining sunitinib with molecules that prevent lysosomal accumulation or inhibit the proteasome.

The BMI1 polycomb protein regulates self-renewal, proliferation and survival of cancer-initiating cells essentially through epigenetic repression of the CDKN2A tumor suppressor locus. We demonstrate here for the first time that BMI1 also prevents autophagy in chronic myeloid leukemia (CML) cell lines, to support their proliferation and clonogenic activity. Using chromatin immunoprecipitation, we identified CCNG2/cyclin G2 (CCNG2) as a direct BMI1 target. BMI1 downregulation in CD34+ CML cells by PTC-209 pharmacological treatment or shBMI1 transduction triggered CCNG2 expression and decreased clonogenic activity. Also, ectopic expression of CCNG2 in CD34+ CML cells strongly decreased their clonogenicity. CCNG2 was shown to act by disrupting the phosphatase 2A complex, which activates a PKCζ-AMPK-JNK-ERK pathway that engages autophagy. We observed that BMI1 and CCNG2 levels evolved inversely during the progression of CML towards an acute deadly phase, and therefore hypothesized that BMI1 could support acute transformation of CML through the silencing of a CCNG2-mediated tumor-suppressive autophagy response.

Deregulated expression of glycolytic enzymes contributes not only to the increased energy demands of transformed cells but also has non-glycolytic roles in tumors. However, the contribution of these non-glycolytic functions in tumor progression remains poorly defined. Here, we show that elevated expression of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), but not of other glycolytic enzymes tested, increased aggressiveness and vascularization of non-Hodgkin's lymphoma. Elevated GAPDH expression was found to promote nuclear factor-κB (NF-κB) activation via binding to tumor necrosis factor receptor-associated factor-2 (TRAF2), enhancing the transcription and the activity of hypoxia-inducing factor-1α (HIF-1α). Consistent with this, inactive mutants of GAPDH failed to bind TRAF2, enhance HIF-1 activity or promote lymphomagenesis. Furthermore, elevated expression of gapdh mRNA in biopsies from diffuse large B-cell non-Hodgkin's lymphoma patients correlated with high levels of hif-1α, vegf-a, nfkbia mRNA and CD31 staining. Collectively, these data indicate that deregulated GAPDH expression promotes NF-κB-dependent induction of HIF-1α and has a key role in lymphoma vascularization and aggressiveness.

Dear Editor, REDD1 (regulated in development and DNA damage responses 1; DDIT4) is a 232 amino-acid TSC2 activator and mTORC1-dependent signaling pathway inhibitor.1, 2 Although REDD1 expression has been characterized in response to hypoxia, DNA damage, and energy deprivation,1, 3, 4 recent studies highlight new posttranslational mechanisms bearing on REDD1 stability.5, 6 To identify additional modifications contributing to REDD1 maintenance, we selected two human acute T-cell leukemia and chronic myeloid leukemia lines (JA3 and K562, respectively) expressing high endogenous REDD1 levels, and exposed both to pro- and anti-proliferative stimuli. Pro-apoptotic inducers Fas receptor activator (CH11) in JA3 cells (Figure 1a, left panel) and imatinib in K562 cells (Figure 1a, middle panel) decreased REDD1 protein levels and promoted accumulation of multiple lower bands detected around 17 kDa. V5-tagged REDD1-overexpressing HeLa cells treated with apoptosis-inducing ligand TRAIL clarified the accumulation of a triplet of bands at 17 kDa, identifiable as products of REDD1–V5 cleavage due to V5-directed antibody recognition (Figure 1a, right panel). Similar to procaspase 3 and caspase 3 substrate protein PARP, a positive control for apoptosis induction, REDD1 protein level remained unchanged in the presence of pan-caspase inhibitor zVAD-fmk, an effect observed in combination with each of the apoptosis-inducing agents tested, suggesting that caspases may directly cleave REDD1 during apoptotic activation. Figure 1 (a, left panel) Western blot for indicated proteins on lysates from JA3 cells co-treated for 8 h with various concentrations of anti-human Fas-activating antibody, CH11 (Millipore, Billerica, MA, USA), and 50 μM pan-caspase inhibitor, ... To test this hypothesis, REDD1-encoding cDNA was transcribed/translated in vitro and the product of this reaction was incubated with recombinant caspases 3, 6, and 7. Only recombinant caspase 3 fully reproduced in a dose-dependent manner the pattern of REDD1 cleavage observed in intact cells, an effect blocked by zVAD-fmk (Figure 1b, upper left panel). We next mutated those REDD1 aspartate residues most conserved across species into non-cleavable alanines to identify sites directly targeted by caspase 3 (Figure 1b, lower left panel). Whereas REDD1 D58A, D74A, and D80A were partially resistant to caspase 3-mediated REDD1 cleavage, the triple mutant (TM, D58A/D74A/D80A) exhibited complete resistance, suggesting that caspase 3 cleaves REDD1 at each of these residues. To assess the functional effects of this cleavage, we generated truncated forms of REDD1 corresponding to the cleavage products (Figure 1b, right panel). Only REDD1 ΔNter1 was stable following overexpression in cells; the others were readily degraded by the proteasome (not shown). Given the known function of REDD1 on mTOR pathway regulation, we overexpressed wild-type (WT), TM, and ΔNter1 REDD1 in HeLa cells exposed to brief amino-acid starvation or treated with insulin. As determined by phosphorylation levels of two mTOR downstream targets, p70S6K and RPS6, REDD1 WT and TM inhibited mTOR pathway, an effect enhanced upon insulin stimulation; REDD1 ΔNter1, however, lost the ability to modulate mTOR pathway activation (Figure 1c, left panel). Colony formation assays revealed that REDD1 ΔNter1 also conferred the highest anti-clonogenic potential (Figure 1c, right panel). Apart from known regulatory effects on the mTOR pathway, REDD1 cleavage by caspase 3 therefore unmasks a new role for REDD1 in cell survival alteration. Recent findings suggest that REDD1 may interact directly with mitochondrial metabolism, as endogenous REDD1 can localize to mitochondria.7 We found that overexpression of REDD1 ΔNter1 rendered HeLa cells more sensitive to low doses of TRAIL and staurosporine (not shown), suggesting that cleaved REDD1 ΔNter1 may alter mitochondrial pathways to promote drastic anti-proliferative properties and, within the context of apoptosis induction, act as a pro-apoptotic amplification signal. Moreover, in and ex vivo studies show that Redd1 knockout cells undergo a more pronounced apoptosis than Redd1 WT cells in response to dexamethasone or doxorubicin,3, 8 confirming that anti-apoptotic function is dampened by caspase 3-mediated destabilization. The REDD1 ΔNter1 product not only marks the loss of REDD1 anti-apoptotic function, but may also act as a trigger that itself initiates robust deleterious effects.

In pathological conditions, the amount of DJ-1 determines whether a cell can survive or engage a cell death program. This is exemplified in epithelial cancers, in which DJ-1 expression is increased, while autosomal recessive early onset Parkinson's disease mutations of DJ-1 generally lead to decreased stability and expression of the protein. We have shown previously that DJ-1 is cleaved by caspase-6 during induction of apoptosis. We demonstrate here that the N-terminal cleaved fragment of DJ-1 (DJ-1 Nt) is specifically expressed in the nucleus and promotes apoptosis in SH-SY5Y neuroblastoma cell lines. In addition, overexpression of DJ-1 Nt in different cell lines leads to a loss of clonogenic potential and sensitizes to staurosporin and 1-methyl-4-phenylpyridinium (MPP+)-mediated caspase activation and apoptosis. Importantly, inhibition of endogenous DJ-1 expression with sh-RNA or DJ-1 deficiency mimics the effect of DJ-1 Nt on cell growth and apoptosis. Moreover, overexpression of DJ-1 Nt increases reactive oxygen species (ROS) production, and sensitizes to MPP+-mediated apoptosis and DJ-1 oxidation. Finally, specific exclusion of DJ-1 Nt from the nucleus abrogates its pro-apoptotic effect. Taken together, our findings identify an original pathway by which generation of a nuclear fragment of DJ-1 through caspase 6-mediated cleavage induces ROS-dependent amplification of apoptosis.

Normothermic liver ischemia-reperfusion (I-R) may induce hepatocellular autophagy, apoptosis, and necrosis. The aim of this study was to investigate these three types of cell death in normothermic liver I-R in rats. A segmental normothermic ischemia of the liver was induced for 120 minutes. Liver autophagy was evaluated by transmission electron microscopy and LC3 (Light Chain 3) immunohistochemical studies. Liver apoptosis was assessed by FLIVO (FLuorescence in vIVO) and TUNEL (TdT-mediated dUTP nick end labeling) assays. Liver necrosis was determined by optical microscopic examination. Autophagy was increased in ischemic liver lobes at 6 hours after reperfusion, compared with nonischemic lobes. Fluorescence microscopy showed in situ caspase-3 and -7 specific activity to be increased in ischemic liver lobes after 6 hours of reperfusion, compared with nonischemic lobes. Quantitative analysis of apoptotic cells evaluated by the TUNEL method showed a clearly significant increase in ischemic liver lobes at 6 hours after reperfusion, compared with nonischemic lobes. Necrotic cell death was significantly increased in ischemic liver lobes at 6 hours after reperfusion, compared with nonischemic lobes (P < .005). In conclusion, 120 minutes normothermic liver I-R resulted in increased autophagic, apoptotic and necrotic cell death.

Imatinib is the leading compound to treat patients with chronic myelogenous leukemia (CML) but the exact mechanism of its anti-leukemic effect is incompletely elucidated. Through inhibition of BCR–ABL, Imatinib blocks several downstream pathways and induces apoptosis of BCR–ABL positive cells. In this study, we analyzed further the mode of action of Imatinib in different appropriate cellular models of CML either sensitive or resistant to Imatinib and in CD34+ cells from CML patients. Pharmacological or short hairpin RNA-mediated inhibition of BCR–ABL triggers lysosomal membrane permeabilization (LMP) that culminates in activation and redistribution of Cathepsin B (CB) into the cytoplasm of CML cells, in which it triggers directly BCR–ABL degradation. Pharmacological inhibition of CB by CA-074Me or small interfering RNA-mediated knock-down of CB partly protects K562 cells from Imatinib-induced cell death and CB overexpression sensitizes these cells to Imatinib killing. Strikingly, Imatinib-triggered LMP, CB activation and BCR–ABL cleavage in CD34+ cells from CML patients and inhibition of CB confers protection against cell death in clonogenic assays of CD34+ primary cells from CML patients. Hence, we describe an original pathway by which Imatinib participates to the elimination of CML cells through LMP and CB-mediated specific degradation of BCR–ABL.

Once cleaved by caspases, the Lyn tyrosine kinase (LynΔN) is relocalized from the plasma membrane to the cytoplasm of apoptotic cells, but the function of such a cleavage is incompletely understood. We evaluated the effect of LynΔN overexpression on imatinib sensitivity of the chronic myelogenous leukemia (CML) cell line K562. Therefore, we generated stable cells that express plasmids encoding LynΔN or its catalytically inactive counterpart LynΔNKD. We established that Lyn is cleaved in imatinib-treated parental K562 cells in a caspase-dependent manner. Lyn cleavage also occurred following BCR-ABL silencing by specific short hairpin RNA (sh-RNA). Imatinib-induced apoptosis was abrogated in LynΔN-overexpressing cells, but not in cells overexpressing its inactive counterpart. Conversely, the overexpression of LynΔN failed to affect the differentiation of K562 cells. Importantly, the protective effect of LynΔN was suppressed by two inhibitors of Lyn activity. LynΔN also inhibits imatinib-mediated caspase-3 activation in the small proportion of nilotinib-resistant K562 cells overexpressing Lyn that can engage an apoptotic program upon imatinib stimulation. Finally, Lyn knockdown by sh-RNA altered neither imatinib-mediated apoptosis nor differentiation. Taken together, our data show that the caspase-cleaved form of Lyn exerts a negative feedback on imatinib-mediated CML cell apoptosis that is entirely dependent on its kinase activity and likely on the BCR–ABL pathway.

NF-κB interferes with the effect of most anti-cancer drugs through induction of anti-apoptotic genes. Targeting NF-κB is therefore expected to potentiate conventional treatments in adjuvant strategies. Here we used a pharmacological inhibitor of the IKK2 kinase (AS602868) to block NF-κB activation. In human colon cancer cells, inhibition of NF-κB using 10 μ M AS602868 induced a 30–50% growth inhibitory effect and strongly enhanced the action of SN-38, the topoisomerase I inhibitor and CPT-11 active metabolite. AS602868 also potentiated the cytotoxic effect of two other antineoplasic drugs: 5-fluorouracil and etoposide. In xenografts experiments, inhibition of NF-κB potentiated the antitumoural effect of CPT-11 in a dose-dependent manner. Eighty-five and 75% decreases in tumour size were observed when mice were treated with, respectively, 20 or 5 mg kg−1 AS602868 associated with 30 mg kg−1 CPT-11 compared to 47% with CPT-11 alone. Ex vivo tumour analyses as well as in vitro studies showed that AS602868 impaired CPT-11-induced NF-κB activation, and enhanced tumour cell cycle arrest and apoptosis. AS602868 also enhanced the apoptotic potential of TNFα on HT-29 cells. This study is the first demonstration that a pharmacological inhibitor of the IKK2 kinase can potentiate the therapeutic efficiency of antineoplasic drugs on solid tumours.

Normothermic liver ischemia-reperfusion (I-R) may induce hepatocellular apoptosis. Caspase activation is involved in the initiation and execution of apoptosis. The aim of this study was to determine in vivo caspase activity in normothermic liver I-R in rats. Segmental normothermic ischemia of the liver was induced for 120 minutes in rats. After intravenous injection of the green probe FLIVO, in vivo caspase-3- and -7-specific activity was determined using fluorescence microscopy, in either nonischemic or ischemic liver lobes at 3 and 6 hours after reperfusion. Liver apoptosis was assessed by the deoxynucleotide transferase-mediated deoxyuridine triphosphate nick end labeling (TUNEL) assay. Fluorescence microscopy showed that in vivo caspase-3- and -7-specific activities were significantly increased (P< .005) in ischemic lobes at 3 and 6 hours of reperfusion, compared with nonischemic liver lobes. Quantitative analysis of apoptotic cells measured by the TUNEL method showed a significant increase among apoptotic cells in ischemic lobes at 3 and 6 hours after reperfusion (P< .005), compared with nonischemic liver lobes. In conclusion, 120-minute normothermic liver I-R resulted in increased caspase-3- and -7-specific activities and in liver cell apoptosis.

Metformin is a widely used antidiabetic agent, which regulates glucose homeostasis through inhibition of liver glucose production and an increase in muscle glucose uptake. Recent studies suggest that metformin may reduce the risk of cancer, but its mode of action in cancer remains not elucidated. We investigated the effect of metformin on human prostate cancer cell proliferation in vitro and in vivo. Metformin inhibited the proliferation of DU145, PC-3 and LNCaP cancer cells with a 50% decrease of cell viability and had a modest effect on normal prostate epithelial cell line P69. Metformin did not induce apoptosis but blocked cell cycle in G(0)/G(1). This blockade was accompanied by a strong decrease of cyclin D1 protein level, pRb phosphorylation and an increase in p27(kip) protein expression. Metformin activated the AMP kinase pathway, a fuel sensor signaling pathway. However, inhibition of the AMPK pathway using siRNA against the two catalytic subunits of AMPK did not prevent the antiproliferative effect of metformin in prostate cancer cells. Importantly, oral and intraperitoneal treatment with metformin led to a 50 and 35% reduction of tumor growth, respectively, in mice bearing xenografts of LNCaP. Similar, to the in vitro study, metformin led to a strong reduction of cyclin D1 protein level in tumors providing evidence for a mechanism that may contribute to the antineoplastic effects of metformin suggested by recent epidemiological studies.

La metformine est un médicament couramment prescrit contre le diabète de type II, qui inhibe la production hépatique de glucose et augmente la captation de glucose par les muscles. Des études épidémiologiques ont montré que la metformine réduit le risque d'apparition de certains cancers. Cependant, son mode d'action sur les cellules cancéreuses reste inconnu. Nous avons étudié l'action de la metformine sur la croissance des lignées cancéreuses de la prostate humaine (DU145, PC-3, LNCaP) in vitro et in vivo. La metformine est un activateur de la voie AMPK (AMP activated Protein Kinase), un senseur de l'énergie cellulaire, celle-ci conduit à l'inhibition de mTOR connue pour induire la prolifération cellulaire et la synthèse protéique. Nous montrons que la metformine n'induit pas l'apoptose mais un arrêt du cycle cellulaire en phase G0/G1 dans les cellules cancéreuses de prostate humaine. Cet arrêt s'accompagne par une forte diminution de la cycline D1 et de la phosphorylation de pRb mais également par une augmentation de l'expression de p27. Bien que la Metformine active la voie AMPK, celle-ci ne semble pas impliquée dans cet effet anti-prolifératif. En effet, l'inhibition de la voie AMPK à l'aide d'ARN interférent ne prévient pas l'effet de la metformine sur la croissance des cellules cancéreuses de la prostate. De façon intéressante, la metformine administrée par voie orale ou intrapéritonéale conduit à une diminution importante et significative de la croissance tumorale dans un modèle de xénogreffe chez la souris nude. De plus, comme observé in vitro, la metformine entraîne une diminution du niveau d'expression de la cycline D1 dans les tumeurs. Nous montrons que la metformine exerce un effet anti-néoplasique qui passe par un arrêt du cycle cellulaire. Ce travail pourrait ainsi expliquer les résultats observés par les récentes études épidémiologiques.

Imatinib targets the Bcr-Abl oncogene that causes chronic myelogenous leukemia (CML) in humans. Recently, we demonstrated that besides triggering apoptosis in K562 cells, imatinib also mediated their erythroid differentiation. Although both events appear to proceed concomitantly, it is not known at present whether or not imatinib-induced apoptosis and differentiation are interdependent processes. Hence, we investigated the requirements for Bcr-Abl inhibitor-mediated apoptosis and erythroid differentiation in several established and engineered CML cell lines. Imatinib triggered apoptosis and erythroid differentiation of different CML cell lines, but only apoptosis exhibited sensitivity to ZVAD-fmk inhibition. Conversely, the p38 mitogen-activated protein (MAP) kinase inhibitor, SB202190, significantly slowed down erythroid differentiation without affecting caspase activation. Furthermore, imatinib and PD166326, another Bcr-Abl inhibitory molecule, triggered erythroid differentiation of K562 cell clones, nevertheless resistant to Bcr-Abl inhibitor-induced apoptosis. Finally, short hairpin RNA inhibitor (shRNAi) silencing of caspase 3 efficiently inhibited caspase activity but had no effect on erythroid differentiation, whereas silencing of Bcr-Abl mimicked imatinib or PD166326 treatment, leading to increased apoptosis and erythroid differentiation of K562 cells. Taken together, our findings not only demonstrate that Bcr-Abl inhibitor-mediated apoptosis and differentiation are fully distinguishable events, but also that caspases are dispensable for erythroid differentiation of established CML cell lines.

The K562 cell line serves as a model to study the molecular mechanisms associated with leukemia differentiation. We show here that cotreatment of K562 cells with PMA and low doses of SB202190 (SB), an inhibitor of the p38 MAPK pathway, induced a majority of cells to differentiate towards the megakaryocytic lineage. Electronic microscopy analysis showed that K562 cells treated with PMA+SB exhibited characteristic features of physiological megakaryocytic differentiation including the presence of vacuoles and demarcation membranes. Differentiation was also accompanied by a net increase in megakaryocytic markers and a reduction of erythroid markers, especially when both effectors were present. PMA effect was selectively mediated by new PKC isoforms. Differentiation of K562 cells by the combination of PMA and SB required Erk1/2 activation, a threshold of JNK activation and p38 MAPK inhibition. Interestingly, higher concentrations of SB, which drastically activated JNK, blocked megakaryocytic differentiation, and considerably increased cell death in the presence of PMA. c-DNA microarray membranes and PCR analysis allow us to identify a set of genes modulated during PMA-induced K562 cell differentiation. Several gene families identified in our screening, including ephrins receptors and some angiogenic factors, had never been reported so far to be regulated during megakaryocytic differentiation.

Abstract The regulation of the phospholipase C (PLC) and the expression of inositol 1,4,5-trisphosphate receptors (IP3Rs) in terms of mRNA, proteins, and binding capacity were examined in the rat myometrium and endometrium at midgestation (Day 12) and at term (Day 21) comparatively to the estrogen-treated tissues (Day 0). In both uterine tissues, the production of inositol phosphates mediated by carbachol as well as by AlF4− was enhanced with advancing gestation. 3[H]IP3 binding sites in membranes also increased during pregnancy (Day 21 > Day 12 > Day 0). The mRNAs encoding for three isoforms of IP3R as well as their corresponding proteins, IP3R-1, IP3R-2, and IP3R-3 were coexpressed, albeit to different extents, in the myometrium and endometrium. The expression of IP3Rs increased with advancing gestation, except for IP3R-2 that increased only in the endometrium at term. Thus, the pregnancy-related upregulation of the PLC cascade coincided with an increase in the expression of IP3Rs. The difference noted between the two uterine tissues suggests that IP3Rs may have cell-specific functions.

The regulation of the phospholipase C (PLC) and the expression of inositol 1,4,5-trisphosphate receptors (IP(3)Rs) in terms of mRNA, proteins, and binding capacity were examined in the rat myometrium and endometrium at midgestation (Day 12) and at term (Day 21) comparatively to the estrogen-treated tissues (Day 0). In both uterine tissues, the production of inositol phosphates mediated by carbachol as well as by AlF4- was enhanced with advancing gestation. (3)[H]IP3 binding sites in membranes also increased during pregnancy (Day 21 > Day 12 > Day 0), The mRNAs encoding for three isoforms of IP3R as well as their corresponding proteins, IP3R-1, IP3R-2, and IP3R-3 were coexpressed, albeit to different extents, in the myometrium and endometrium. The expression of IP(3)Rs increased with advancing gestation, except for IP3R-2 that increased only in the endometrium at term. Thus, the pregnancy-related upregulation of the PLC cascade coincided with an increase in the expression of IP(3)Rs. The difference noted between the two uterine tissues suggests that IP(3)Rs may have cell-specific functions.

The study of epidemiology and of the carcinogenesis in epidermoid carcinomas of the upper aerodigestive tract shows that their occurrence is not random. Tobacco abuse plays a major role, especially because of benzopyrene, mutagen of the P53 gene, however it is associated with many other potentiating factors: alcohol, metals, hydrocarbures, virus, food, climate, genetic fragility that create genetic lesions at the origin of carcinogenesis. The latter occurs as "field cancerization" with multiple alterations of the mucosa and general attack of the control systems of the differentiation, growth and cell apoptosis which usually protect the cell against the phenomena of carcinogenesis. The P53 protein gene, retinoid receptors as well as the system of detoxifying glutathion S transferase are modified at the very early stage of these diseases, these abnormalities can be logically related to epidemiological data. These data lead us therefore to imagine complementary specific reverting therapies of induced genetic abnormalities, through the reexpression of non mutated gene encoding P53 protein and the use of retinoid. These various modalities are reported hereafter.

The influence of antibiotics, particularly ofloxacin (OF), a commonly used antimicrobial fluoroquinolone, on the multidrug resistance (MDR) phenotype of the HCT-8 cell line was studied. This cell line was grown in OF containing medium for several months and the expression of the MDR phenotype was followed through the analysis of the expression and functionality of the P-glycoprotein (Pgp), the chemosensitivity to daunorubicin (DNR), and the mRNA expression of mdr-1, multidrug resistance-associated protein (MRP), and topoisomerase IIalpha and IIbeta genes. Replacement of OF by penicillin streptomycin (PS) resulted in a significant decrease in mdr-1 mRNA expression, which was found to correlate with a decrease in the expression and functionality of the Pgp. After antibiotic starvation for 4 weeks, cells grown in antibiotic-free medium were then exposed to PS or OF; these cells showed an increase in mdr-1 mRNA/Pgp and MRP mRNA expression without a decrease in DNR cytotoxicity. OF cultured cells exhibited a significant increase in Pgp expression without evidence of the functionality of the Pgp. An increase in topoisomerase IIalpha mRNA expression was observed with time and with the number of passages of the cell line without any relationship to the presence of antibiotics in the culture medium. These results showed that extensive use of antibiotics, particularly the quinolones, can modify the phenotype of the HCT-8 colon adenocarcinoma cell line.

The multidrug resistance (MDR) modulating activity of SDZ‐PSC833 (PSC), a non‐immunosuppressive cyclosporine analogue, was investigated and compared with cyclosporin A (CSA) in bone marrow clinical specimens from 45 patients with acute myeloid leukaemia (AML) taken at diagnosis, using double‐labelling flow cytometry with simultaneous determination of P‐glycoprotein (PGP) expression and intracellular daunorubicin fluorescence (IDF). On the basis of pre‐clinical results in multidrug‐resistant K562 leukaemic cells, concentrations leading to iso‐effective complete restoration of IDF were used: 5 and 10 μmol/l, respectively for PSC and CSA. In the clinical specimens, PGP expression was correlated with a significant decrease in IDF. PSC was found to be significantly more potent than CSA since it was found to induce a significant increase in IDF in a higher number of cases and to a higher extent than CSA. PGP‐unrelated activity of PSC was also observed in specimens expressing no PGP but exhibiting low IDF, thus probably expressing alternative resistance mechanisms. The results confirm the potency of PSC as MDR‐modulating agent in clinical AML specimens whose resistance pattern differed from that of highly resistant cell models and suggest that the activity of PSC is not limited to P‐glycoprotein inhibition.