Biomedical subjects

S Spinelli

Publications and source records attributed to S Spinelli.

At least 37 records · Page 2Linked to original sources

PubMed2000

Structural basis of sialyltransferase activity in trypanosomal sialidases.

The intracellular parasite Trypanosoma cruzi, the etiological agent of Chagas disease, sheds a developmentally regulated surface trans-sialidase, which is involved in key aspects of parasite-host cell interactions. Although it shares a common active site architecture with bacterial neuraminidases, the T.cruzi enzyme behaves as a highly efficient sialyltransferase. Here we report the crystal structure of the closely related Trypanosoma rangeli sialidase and its complex with inhibitor. The enzyme folds into two distinct domains: a catalytic beta-propeller fold tightly associated with a lectin-like domain. Comparison with the modeled structure of T.cruzi trans-sialidase and mutagenesis experiments allowed the identification of amino acid substitutions within the active site cleft that modulate sialyltransferase activity and suggest the presence of a distinct binding site for the acceptor carbohydrate. The structures of the Trypanosoma enzymes illustrate how a glycosidase scaffold can achieve efficient glycosyltransferase activity and provide a framework for structure-based drug design.

Animals↗

PubMed2000

Blockade of latent inhibition following pharmacological increase or decrease of GABA(A) transmission.

The latent inhibition (LI) phenomenon refers to the retardation in learning of an association between a stimulus and a consequence if that stimulus had been previously experienced without consequence. An earlier study demonstrated that the benzodiazepine receptor agonist chlordiazepoxide (CDP), when administered before the phase of preexposure to the to-be-conditioned stimulus, impaired animals' ability to develop LI. The present study was designed to investigate the effect of the anxiogenic drugs pentylenetetrazole (PTZ) and the benzodiazepine partial inverse agonist Ro15-4513 on LI. Both anxiogenics, in contrast to CDP, are known for their GABA inhibitory action. The effects produced by the combined administration of a GABAergic function facilitator and inhibitor (CDP/PTZ and CDP/Ro15-4513) were also investigated. Both anxiogenic drugs led to an attenuation of LI, and, similarly to CDP, this attenuation was exclusively due to their administration prior to the preexposure stage of the experiment. However, this effect was abolished when anxiolytic and anxiogenic drugs were administered together, suggesting a pharmacological rather than behavioral summation of effects. These data also demonstrate the bidirectional GABAergic modulation of the LI phenomenon: both increased and decreased GABA(A) receptor activation led to reduced LI, thereby suggesting that an optimal receptor activation level is necessary for the normal establishment of LI.

Affinity Labels↗

PubMed2000

The effects of ibotenic acid lesions of the medial and lateral prefrontal cortex on latent inhibition, prepulse inhibition and amphetamine-induced hyperlocomotion.

Hypofunction of prefrontal cortical regions, such as dorsolateral and orbital regions, has been suggested to contribute to the symptomatology of schizophrenia. In the rat, the medial and the lateral prefrontal cortices are considered as homologs of the primate dorsolateral and orbital prefrontal cortices, respectively. The present study investigated in rats the effects of lesions of the medial and lateral prefrontal cortices on latent inhibition, prepulse inhibition and amphetamine-induced activity. These paradigms are known to be modulated by the mesolimbic dopaminergic system, a system that has been suggested to be involved in the symptomatology of schizophrenia. Latent inhibition and prepulse inhibition are disrupted in schizophrenic patients as well as in rats treated with amphetamine. Amphetamine-induced activity was tested under dim light (low stress) and bright light (high stress) because stressful situations selectively increase mesocortical dopamine activity. Lateral prefrontal cortex lesioned animals did not differ in their behavior from control animals in any of the paradigms used in this study. Medial prefrontal cortex lesions did not affect latent inhibition but increased prepulse inhibition. In the amphetamine-induced activity experiment, prior to drug administration, open field locomotion was reduced under bright illumination for all lesion groups. After amphetamine administration, medial prefrontal cortex lesions attenuated the hyperlocomotor effect of the drug under the dim light condition and potentiated it under the bright light condition. The results indicate that medial and lateral prefrontal cortex can be functionally differentiated by their involvement in the modulation of behavior requiring mesocorticolimbic dopamine activation. The results in amphetamine induced activity suggest that the behavioral outcomes associated with medial prefrontal cortex depend on the background (stress) against which the evaluation is made. The results also support the notion that prepulse inhibition may be a better model than latent inhibition of the symptoms of schizophrenia associated with dysfunctional prefrontal activity.

Amphetamine↗

PubMed2000

Differential role of the medial and lateral prefrontal cortices in fear and anxiety.

In the rat, both the medial and lateral prefrontal cortices (PFC; mPFC and lPFC, respectively) have direct connections with limbic structures that are important in the expression of fear and anxiety. The present study investigated the behavioral effects of excitotoxic lesions of either the mPFC or the lPFC on conditioned and unconditioned fear paradigms. In both unconditioned fear paradigms (open field, elevated plus-maze), lesions of the mPFC decreased anxiety. In fear conditioning, lPFC lesions substantially increased freezing throughout the different phases of the experiment, whereas mPFC lesions increased freezing to contextual cues and showed reduced freezing to discrete cues. These results support the functional role of the PFC in mediating or modulating central states of fear and anxiety and suggest a functional dissociation between the lPFC and mPFC in their role in fear and anxiety.

Animals↗

PubMed2000

Quality of life in 244 recipients of allogeneic bone marrow transplantation.

The number of long-term survivors after allogeneic bone marrow transplantation (BMT) has been increasing over the past years, and quality of life (QOL) has become an important end-point. We studied 244 patients undergoing an allogeneic BMT to identify factors and events influencing psychosocial outcome. Patients enrolled received the Psychosocial Adjustment to Illness Scale (PAIS) questionnaire assessing psychological and social adjustment to chronic illness or its sequelae. Eighty-two per cent of patients had a haematological disease. The median age was 28 years at BMT, and the median follow-up was 61 months. The median overall PAIS score for all patients was 56 (range 22-76): 25% (n = 61) of patients were considered to have a good QOL (</= 25 percentile score); 44% (n = 108) of patients had an intermediate QOL (26-75 percentile score) and 31% (n = 75) had a poor QOL (> 75 percentile score). Factors associated with a poor QOL in multivariate analysis were: patients' age at BMT (> 25 years, P < 0.01); presence of long-term sequelae (P < 0.01); chronic graft-versus-host disease (GVHD) (P < 0.05); and a short interval from BMT (< 5 years; P < 0.05). The QOL improved with time: 12% of patients reported a good QOL within 5 years compared with 38% after this time point and, conversely, 38% reported a poor QOL within 5 years compared with 24% after this time point (P < 0. 0001). Older patients had significantly poorer QOL compared with younger patients (< or = 25 years; P = 0.01). Females had significantly poorer scores when compared with males in the sexual (P < 0.0001) and psychological domains (P = 0.001). The data suggest that (i) one-third of patients undergoing allogeneic BMT report a poor QOL; (ii) factors associated with poor QOL are older age, presence of long-term sequelae, chronic GVHD and short follow-up; (iii) QOL is superior in long-term survivors; and (iv) BMT affects different aspects of life in males and females. A longitudinal study is ongoing to prove the effect of time on quality of life.

Adolescent↗

PubMed1999

Insight into odorant perception: the crystal structure and binding characteristics of antibody fragments directed against the musk odorant traseolide.

Monoclonal antibodies were elicited against the small hydrophobic hapten traseolide, a commercially available musk fragrance. Antibody variable region sequences were found to belong to different sequence groups, and the binding characteristics of the corresponding antibody fragments were investigated. The antibodies M02/01/01 and M02/05/01 are highly homologous and differ in the binding pocket only at position H93. M02/05/01 (H93 Val) binds the hapten traseolide about 75-fold better than M02/01/01 (H93 Ala). A traseolide analog, missing only one methyl group, does not have the characteristic musk odorant fragrance. The antibody M02/05/01 binds this hapten analog about tenfold less tightly than the original traseolide hapten, and mimics the odorant receptor in this respect, while the antibody M02/01/01 does not distinguish between the analog and traseolide. To elucidate the structural basis for the fine specificity of binding, we determined the crystal structure of the Fab fragment of M02/05/01 complexed with the hapten at 2.6 A resolution. The crystal structure showed that only van der Waals interactions are involved in binding. The somatic Ala H93 Val mutation in M02/05/01 fills up an empty cavity in the binding pocket. This leads to an increase in binding energy and to the ability to discriminate between the hapten traseolide and its derivatives. The structural understanding of odorant specificity in an antibody gives insight in the physical principles on how specificity for such hydrophobic molecules may be achieved.

Amino Acid Sequence↗

PubMed1999

Crystal structure of a ternary complex between human chorionic gonadotropin (hCG) and two Fv fragments specific for the alpha and beta-subunits.

Human chorionic gonadotropin (hCG), is a placental hormone which exerts its major effect by stimulating progesterone production, crucially sustaining the early weeks of pregnancy. Detection of hCG with specific monoclonal antibodies (mAbs) has become the chosen means for pregnancy diagnosis. We have used antibody Fv fragments derived from two high-affinity mAbs, one against the alpha and the other against the beta-hCG subunit to enable the crystallisation of intact or desialylated hCG. Crystals of a ternary complex composed of Fv anti-alpha/hCG/Fv anti-beta were found to diffract to 3.5 A resolution, and the structure was solved by molecular replacement. In the crystal, the two Fvs keep hCG as in a molecular cage, providing good protein-protein contacts and leaving enough space for the saccharides to be accommodated in the cell solvent. The two Fvs were found not to interact directly through their complementary-determining regions with the hCG saccharides, but only with the protein. The hCG structure in the ternary complex was very close to that of the HF partially deglycosylated hormone, thus indicating that neither the saccharides nor the Fvs had any substantial influence on hormone structure.

Amino Acid Sequence↗

PubMed1999

The cellular basis of the efficacy of the trinuclear platinum complex BBR 3464 against cisplatin-resistant cells.

Multinuclear platinum compounds have been designed to circumvent the cellular resistance to conventional mononuclear platinum-based drugs. In this study we performed a comparative study of cisplatin and of the triplatinum complex BBR 3464 in a human osteosarcoma cell system (U2-OS) including an in vitro selected cisplatin-resistant subline (U2-OS/Pt). BBR 3464 was extremely potent in comparison with cisplatin in U2-OS cells and completely overcame resistance of U2-OS/Pt cells. In both cell lines, BBR 3464 accumulation and DNA-bound platinum were higher than those observed for cisplatin. On the contrary, a low frequency of interstrand cross-links after exposure to BBR 3464 was found. Differently from the increase of DNA lesions induced by cisplatin, kinetics studies indicated a low persistence of interstrand cross-link formation for BBR 3464. Western blot analysis of DNA mismatch repair proteins revealed a marked decrease of expression of PMS2 in U2-OS/Pt cells, which also exhibited microsatellite instability. Studies on DNA mismatch repair deficient and proficient colon carcinoma cells were consistent with a lack of influence of the DNA mismatch repair status on BBR 3464 cytotoxicity. In conclusion, the cytotoxic potency and the ability of the triplatinum complex to overcome cisplatin resistance appear to be related to a different mechanism of DNA interaction (formation of different types of drug-induced DNA lesions) as compared to conventional mononuclear complexes.

Adaptor Proteins, Signal Transducing↗

PubMed1999

A novel charged trinuclear platinum complex effective against cisplatin-resistant tumours: hypersensitivity of p53-mutant human tumour xenografts.

Multinuclear platinum compounds were rationally designed to bind to DNA in a different manner from that of cisplatin and its mononuclear analogues. A triplatinum compound of the series (BBR 3464) was selected for preclinical development, since, in spite of its charged nature, it was very potent as cytotoxic agent and effective against cisplatin-resistant tumour cells. Anti-tumour efficacy studies were performed in a panel of human tumour xenografts refractory or poorly responsive to cisplatin. The novel platinum compound exhibited efficacy in all tested tumours and an impressive efficacy (including complete tumour regressions) was displayed in two lung carcinoma models, CaLu-3 and POCS. Surprisingly, BBR 3464 showed a superior activity against p53-mutant tumours as compared to those carrying the wild-type gene. The involvement of p53 in tumour response was investigated in an osteosarcoma cell line, SAOS, which is null for p53 and is highly sensitive to BBR 3464, and in the same cells following introduction of the wild-type p53 gene. Thus the pattern of cellular response was investigated in a panel of human tumour cells with a different p53 gene status. The results showed that the transfer of functional p53 resulted in a marked (tenfold) reduction of cellular chemosensitivity to the multinuclear platinum complex but in a moderate sensitization to cisplatin. In addition, in contrast to cisplatin, the triplatinum complex was very effective as an inducer of apoptosis in a lung carcinoma cell line carrying mutant p53. The peculiar pattern of anti-tumour activity of the triplatinum complex and its ability to induce p53-independent cell death may have relevant pharmacological implications, since p53, a critical protein involved in DNA repair and induction of apoptosis by conventional DNA-damaging agents, is defective in several human tumours. We suggest that the peculiar DNA binding properties of the triplatinum complex may contribute to the striking profile of anti-tumour efficacy. Taken together, the available information supports that anti-tumour activity of the novel compound is mediated by a mechanism different from that of conventional platinum complexes, and compounds of this series could represent a new class of promising anti-tumour agents.

Animals↗

PubMed1999

The structure of an entire noncovalent immunoglobulin kappa light-chain dimer (Bence-Jones protein) reveals a weak and unusual constant domains association.

Monoclonal free light chains secreted in immunoproliferative disorders are frequently involved in renal complications, including a specific proximal tubule impairment, Fanconi's syndrome. The latter is characterized in most cases by intracellular crystallization including a light-chain variable-domain fragment which resists lysosomal proteases. Bence-Jones protein (BJP) DEL was isolated from a patient with myeloma-associated Fanconi's syndrome. The crystal structure of this human kappa immunoglobulin light-chain noncovalent dimer was determined using molecular replacement with the structure of molecule REI, as the variable domain, and that of BJP LOC as the constant domain. To our knowledge, DEL is the first complete kappa BJP structure described to date. The R-factor is 20.7% at 2.8 A resolution. The BJP DEL dimer was compared with other light-chain dimers and with Fab fragments with a kappa light chain. Although the domain-folding pattern was similar, the relative positions of the constant domains differed. BJP DEL showed a noncanonical quaternary structural arrangement which may be attributable to the poor CL-CL affinity and lack of an interchain disulfide bridge, combined with the conformational editing effect of the crystal-packing forces. Our results suggest that, in the absence of a disulfide bridge, most BJP CLs are probably mobile in solution. This may explain their high susceptibility to proteases and the absence of naturally occurring crystals for these dimers. Furthermore, these findings of an unusual quaternary structure of an immunoglobulin light-chain association extend our knowledge about the large and highly diverse structures of the immunoglobulin superfamily.

Amino Acid Sequence↗

PubMed1999

A novel trinuclear platinum complex overcomes cisplatin resistance in an osteosarcoma cell system.

Multinuclear platinum compounds have been designed to circumvent the cellular resistance to conventional platinum-based drugs. In an attempt to examine the cellular basis of the preclinical antitumor efficacy of a novel multinuclear platinum compound (BBR 3464) in the treatment of cisplatin-resistant tumors, we have performed a comparative study of cisplatin and BBR 3464 in a human osteosarcoma cell line (U2-OS) and in an in vitro selected cisplatin-resistant subline (U2-OS/Pt). A marked increase of cytotoxic potency of BBR 3464 in comparison with cisplatin in U2-OS cells and a complete lack of cross-resistance in U2-OS/Pt cells were found. A detailed analysis of the cisplatin-resistant phenotype indicated that it was associated with reduced cisplatin accumulation, reduced interstrand cross-link (ICL) formation and DNA platination, microsatellite instability, and reduced expression of the DNA mismatch repair protein PMS2. Despite BBR 3464 charge and molecular size, in U2-OS and U2-OS/Pt cells, BBR 3464 accumulation and DNA-bound platinum were much higher than those observed for cisplatin. In contrast, the frequency of ICLs after exposure to BBR 3464 was very low. The time course of ICL formation after drug removal revealed a low persistence of these types of DNA lesions induced by BBR 3464, in contrast to an increase of DNA lesions induced by cisplatin, suggesting that components of the DNA repair pathway handle the two types of DNA lesions differently. The cellular response of HCT116 mismatch repair-deficient cells was consistent with a lack of influence of mismatch repair status on BBR 3464 cytotoxicity. Because BBR 3464 produces high levels of lesions different from ICLs, likely including intra-strand cross-links and monoadducts, the ability of the triplatinum complex to overcome cisplatin resistance appears to be related to a different mechanism of DNA interaction (formation of different types of drug-induced DNA lesions) as compared with conventional mononuclear complexes rather than the ability to overcome specific cellular alterations.

Antineoplastic Agents↗

PubMed1998

Synthesis and antitumor evaluation of 2,5-disubstituted-indazolo[4, 3-gh]isoquinolin-6(2H)-ones (9-aza-anthrapyrazoles).

The synthesis and antitumor evaluation of 2, 5-disubstituted-indazolo[4,3-gh]isoquinolin-6(2H)-ones (9-aza-APs) are described. The key intermediates in the synthesis are benz[g]isoquinoline-5,10-diones which are substituted at positions 6 and 9 with groups of different nucleofugacity for SNAr displacements. The initial displacement of fluoride by a substituted hydrazine leads to the pyrazole analogues. Substitution of the remaining leaving group by an amine or BOC-protected amines leads to the 9-aza-APs 12. These analogues were converted into their maleate or hydrochloride salts 13. In two cases, namely, 13x and 13z, sidearm buildup was also employed in the synthetic pathway. In vitro evaluation of 9-aza-APs against the human colon tumor cell line LoVo uncovered for most of the compounds a cytotoxic potency lower than that of DuP-941 or mitoxantrone and comparable to that of doxorubicin. Only analogues 13c, 13n, and 13ff were as cytotoxic as DuP-941. Interestingly, while DuP-941 was highly cross-resistant in the LoVo cell line resistant to doxorubicin (LoVo/Dx), the 9-aza-APs carrying a distal lipophilic tertiary amine moiety in both chains were capable of overcoming the MDR resistance induced in this cell line. The 9-aza-APs show outstanding in vivo antitumor activity against both systemic P388 murine leukemia and MX-1 human mammary carcinoma transplanted in nude mice. At their optimal dosages, congeners 13a-c, 13f, 13n, 13q, 13x, and 13dd were highly effective against P388 leukemia with T/C% of 200-381, while the T/C% value of DuP-941 was 147. In the MX-1 tumor model, 24 compounds elicited percentages of tumor weight inhibitions (TWI) ranging from 50% to 99%. Congeners 13d, 13k, 13l, 13x, 13z, and 13ee emerged as the most effective ones, with TWI% 96, simliar to that of DuP-941 (TWI% = 95). On the basis of their efficacy profile in additional experimental tumors and lack of cardiotoxicity in preclinical models, two congeners have surfaced as potential clinical candidates.

Adenocarcinoma↗

PubMed1998

Kinetics and interaction studies between cytochrome c3 and Fe-only hydrogenase from Desulfovibrio vulgaris Hildenborough.

Hydrogenases from Desulfovibrio are found to catalyze hydrogen uptake with low potential multiheme cytochromes, such as cytochrome c3, acting as acceptors. The production of Fe-only hydrogenase from Desulfovibrio vulgaris Hildenborough was improved with respect to the growth phase and media to determine the best large-scale bacteria growth conditions. The interaction and electron transfer from Fe-only hydrogenase to multiheme cytochrome has been studied in detail by both BLAcore and steady-state measurements. The electron transfer between [Fe] hydrogenase and cytochrome c3 appears to be a cooperative phenomenon (h = 1.37). This behavior could be related to the conductivity properties of multihemic cytochromes. An apparent dissociation constant was determined (2 x 10(-7) M). The importance of the cooperativity for contrasting models proposed to describe the functional role of the hydrogenase/cytochrome c3 complex is discussed. Presently, the only determined structure is from [NiFe] hydrogenase and there are no obvious similarities between [NiFe] and [Fe] hydrogenase. Furthermore, no crystallographic data are available concerning [Fe] hydrogenase. The first results on crystallization and X-ray crystallography are reported.

Biosensing Techniques↗

PubMed1998

The structure of the monomeric porcine odorant binding protein sheds light on the domain swapping mechanism.

The X-ray structure of the porcine odorant binding protein (OBPp) was determined at 2.25 A resolution. This lipocalin is a monomer and is devoid of naturally occurring bound ligand, contrary to what was observed in the case of bovine OBP [Tegoni, M., et al. (1996) Nat. Struct. Biol. 3, 863-867; Bianchet, M. A., et al. (1996) Nat. Struct. Biol. 3, 934-939]. In this latter protein, a dimer without any disulfide bridges, domain swapping was found to occur between the beta- and alpha-domains. A single Gly (121) insertion was found in OBPp when it was compared to OBPb, which may prevent domain swapping from taking place. The presence of a disulfide bridge between the OBPp beta- and alpha-domains (cysteines 63 and 155) may lock the resulting fold in a nonswapped monomeric conformation. Comparisons with other OBPs indicate that the two cysteines involved in the OBPp disulfide bridge are conserved in the sequence, suggesting that OBPp may be considered a prototypic OBP fold, and not OBPb.

Amino Acid Sequence↗

PubMed1998

The New York High-Risk Project: social and general intelligence in children at risk for schizophrenia.

UNLABELLED: Social deficits, as well as low performance on intelligence tests, are known early symptoms of schizophrenia. We studied whether impairment of social intelligence can be detected before the outbreak of the disorder. In the New York High-Risk Project, children at risk for schizophrenia (HRSz) or affective disorder (HRAff) and a normal control group (NC) were studied over the past 26 years. The children are now in mid-adulthood, with known psychiatric outcomes. Developmental and clinical data from childhood can now be related to adulthood diagnoses. We compared mean WISC (or WISC-R) and WAIS (or WAIS-R) scores from childhood and adolescence, and change of IQ, between the risk groups, as well as between the adulthood outcomes. We were specifically interested in the development of social intelligence (the Picture Arrangement and Comprehension subtests). We used logistic regression analyses to generate a model predicting adulthood schizophrenia. RESULTS: IQ at age 9,7 was lower in children with HRSz than with HRAff. Adulthood schizophrenia, compared with major depressive disorder and no psychiatric diagnosis could not be related conclusively to low IQ. This may be a result of the study design, since children with IQ below 70 or behavioral problems were not eligible as study subjects. There was no evidence of lower scores or more decline in social intelligence related to age or group membership (risk or outcome). Subtest-Scatter, a nondirectional measure of the differences between all subtests and Vocabulary, reflecting a lesser difference between crystallized and fluid intelligence, was identified as a significant predictor of adulthood schizophrenia, in the whole group as well as in the HRSz group alone.

Adolescent↗

PubMed1998

Sequential annual administration of purified fusion protein vaccine against respiratory syncytial virus in children with cystic fibrosis.

BACKGROUND: We recently showed the clinical benefit of the PFP-2 vaccine for respiratory syncytial virus (RSV) for children with cystic fibrosis (CF). OBJECTIVE: To determine the safety and immunogenicity of yearly sequential administration of the PFP-2 vaccine in CF children. STUDY DESIGN: Twenty-nine of the 34 CF children who participated in the previous study were enrolled in this open label vaccine study. All of the CF children ages 2.6 to 8.9 years received the PFP-2 vaccine, the PFP/PFP group received the PFP-2 vaccine in 1993 and 1994 and the saline/PFP group received the vaccine for the first time in 1994. At entry demographic data and measurements of lung function and nutrition were collected. Microneutralization test, enzyme-linked immunosorbent assay to F protein and Western blot assay were performed on plasma drawn before and 4 weeks after vaccination and at the end of the RSV season. During the study weekly telephone calls were made and acute respiratory illnesses were evaluated. RESULTS: Baseline measurements were similar between groups. Systemic and local vaccine reactions were mild and similar for both groups. A 4-fold or greater neutralizing antibody rise to RSV occurred in 4 of 14 (28.6%) and 9 of 14 (64.3%) in PFP/PFP and saline/PFP groups (P = 0.13), respectively. Four children in the PFP/PFP group and 7 in the saline/PFP group were infected with RSV. A reduction in lower respiratory illnesses (1.0 vs. 2.0), antibiotic courses (2.5 vs. 5.6) and days of illnesses (37.3 vs. 93.1) was observed in the PFP/PFP vaccinees infected with RSV compared with the saline/PFP group (t test; P < or = 0.05). One death occurred in the PFP/PFP group; the cause of death was consistent with septic shock and unrelated to vaccination or RSV infection. CONCLUSION: Sequential annual PFP-2 vaccination was safe and not associated with exaggerated respiratory disease.

Antibodies, Viral↗

PubMed1998

Sensitivity to cisplatin and platinum-containing compounds of Schizosaccharomyces pombe rad mutants.

The role of genes that affect response to radiation in determining sensitivity to platinum-containing compounds was studied using a panel of 23 strains of the yeast Schizosaccharomyces pombe. The radiation-hypersensitive mutants all had the same genetic background and most of them contained mutations that disabled either cell cycle checkpoints or DNA repair. The tested platinum compounds included cisplatin and two complexes containing diaminocyclohexane (oxaliplatin and tetraplatin), two ammine/cyclohexylamine complexes with different orientation of the leaving groups (JM216 and JM335) and a multinuclear platinum complex (BBR 3464). The cytotoxic effect of the selected platinum complexes was evaluated by using a microtiter growth inhibition assay with a 48 hr exposure to drug. The mutants fell into three groups with respect to sensitivity to cisplatin: four mutants (rad2, -7, -11, -15) exhibited minimal change in sensitivity; fifteen mutants (rad4-6, -8-10, -12-14, -16-17, -19-21, and -22) were 5.1-21.7-fold hypersensitive; only rad1 and -3 mutants, defective in checkpoints, and rad18, defective in repair, displayed a marked hypersensitivity. None of the mutants demonstrated appreciable change in sensitivity to JM216 presumably as a consequence of a lack of resistance of the wild-type strain, whereas a moderate increase in sensitivity to JM335 was observed for most of the mutants, and hypersensitivity to BBR3464 was observed only in rad1 and -3. No relevant changes in sensitivity to tetraplatin were observed. Most of the mutants, with the exception of rad2, -7, and -15, were hypersensitive to oxaliplatin. These findings demonstrate that specific mutations have disparate effects on the profile of sensitivity to different members of the same class of cytotoxic agents, which provides genetic evidence that different mechanisms are involved in differential cytotoxicity induced by Pt compounds. The results also demonstrate the utility of such a panel of mutants, constructed on the same genetic background, for detecting specific cellular response; presumably, this reflects the recognition or processing of specific DNA adducts. In conclusion, because the rad1 and rad3 gene products are determinants of cellular response to a large number of platinum-containing compounds, the present results support a critical role of genes involved in cell cycle control in cellular sensitivity to these agents.

Cell Cycle↗

PubMed1997

Physicochemical properties, cytotoxic activity and topoisomerase II inhibition of 2,3-diaza-anthracenediones.

The physicochemical, cytotoxic and pharmacological properties of 2,3-diaza-anthracenedione derivatives were examined to gain insight into the structure-activity relationships in this class of compounds. Spectrophotometric, chiroptical and voltammetric measurements were performed, along with cell cytotoxicity, alkaline elution, topoisomerase II-mediated DNA cleavage and cellular drug-uptake determination. In comparison with classic anthracenediones such as mitoxantrone and ametantrone, the aza derivatives were characterized by less negative reduction potentials, lower affinity for DNA and modified geometry of intercalation. The biological effects of the new compounds were also profoundly affected by bioisosteric N for C replacement. Stimulation of topoisomerase II-mediated DNA cleavage was not observed, whereas other mechanisms of cell cytotoxicity, possibly involving oxidative DNA damage appeared to be operative. The inability to generate protein-associated strand breaks could be explained by an unfavorable orientation of the drug in the intercalation complex rather than by a reduced binding to DNA. Geometry of drug intercalation may have a critical influence on the formation of the ternary complex. In turn, the onset of a different DNA-damaging pathway is likely to be related to easy redox cycling of the 2,3-diaza-substituted anthracenedione derivatives, which could produce radical species to a remarkably greater extent than could the carbocyclic parent drugs.

Anthraquinones↗