Silvio Parodi

Drug metabolism polymorphisms as modulators of cancer susceptibility

Recently, several molecular genetic bases of polymorphic enzyme activities involved in drug activation and detoxification have been elucidated. Many molecular epidemiology studies based on these premises have sought to gather information on the association of genetically determined metabolic variants with different risks of environmentally induced cancer. While rare alterations of tumor suppressor genes dramatically raise cancer risk for the single affected subjects, far more common and less dramatic differences in genes encoding for drug metabolism enzymes can be responsible for a relatively small, but rather frequent increase of cancer risk at the population level. This increase could be especially important in specific cases of occupational, pharmacological or environmental exposure. Examination of the current literature reveals that the most extensively investigated metabolic polymorphisms are those of P450 1A1 and P450 2D6 cytochromes, glutathione S-transferases (GSTs; M1 and, to a lesser extent, M3, P1 and T1) and N-acetyltransferases (NATs; NAT1 and NAT2). Making reference to these enzymes, we have assayed the current knowledge on the relations among polymorphisms of human xenobiotic-metabolizing enzymes and cancer susceptibilities. We have found intriguing models of susceptibility toward different types of cancer. We have reviewed and commented these models on light of the complex balance among different enzyme activities that, in each individual, determines the degree of each cancer susceptibility. Moreover, we have found techniques of molecular genetic analysis, more suitable than previous ones on phenotypic expression, now allowing better means to detect individuals at risk of cancer. According to the models presently available, a systematic screening of individuals at risk seems to make sense only in situations of well defined carcinogenic exposures and when performed by the polymorphism analysis of coordinated enzyme activities concurring to the metabolism of the carcinogen(s) in question. Genetic polymorphism analysis can allow for the detection of patients more prone to some types of specific cancers, or to the adverse effects of specific pharmaceutical agents. Considering the increasingly confirmed double-edged sword nature of metabolism polymorphism (both wild-type and variant alleles can predispose to cancer, albeit in different situations of exposure), individual susceptibility to cancer should be monitored as a function of the nature, and mechanism of action, of the carcinogen(s) to which the individual under study is known to be exposed, and with reference to the main target organ of the considered type of exposure.

Quantitative correlations amongst alkaline DNA fragmentation, DNA covalent binding, mutagenicity in the Ames test and carcinogenicity, for 21 compounds ☆

21 compounds from different chemical classes were quantitatively compared for their carcinogenic potency according to 4 parameters: (1) potency in inducing covalent binding with DNA in vivo; (2) potency in inducing alkaline DNA fragmentation after treatment in vivo; (3) acute toxicity; (4) mutagenic potency in the Ames test. Establishing well-defined conditions for normalization of the different types of data and determination of the set that had to be submitted to statistical analysis appeared to be a difficult task, for which only compromise solutions were possible. A statistical analysis of the data suggested that all parameters considered were correlated with carcinogenic potency. However, we found that there are about 3 chances to 1 that carcinogenicity is better correlated with DNA covalent binding in vivo than it is to mutagenicity in the Ames test. With due precautions, even acute toxicity could be of predictive value. DNA adducts and DNA fragmentation, both in vivo, appeared to be 2 parameters strongly correlated between them. From a multivariate statistical analysis it appeared that: (1) a significant improvement of quantitative predictability is in principle obtainable with a battery of short-term test; and (2) the improvement is obtainable only if the short-term tests considered, while all correlated with carcinogenicity, are relatively independent amongst themselves.

32P‐postlabeling detection of DNA adducts in mice treated with the herbicide roundup

Roundup is a postemergence herbicide acting on the synthesis of amino acids and other important endogenous chemicals in plants. Roundup is commonly used in agriculture, forestry, and nurseries for the control or destruction of most herbaceous plants. The present study shows that Roundup is able to induce a dose‐dependent formation of DNA adducts in the kidneys and liver of mice. The levels of Roundup‐related DNA adducts observed in mouse kidneys and liver at the highest dose of herbicide tested (600 mg/kg) were 3.0 ± 0.1 (SE) and 1.7 ± 0.1 (SE) adducts/108 nucleotides, respectively. The Roundup DNA adducts were not related to the active ingredient, the isopropylammonium salt of glyphosate, but to another, unknown component of the herbicide mixture. Additional experiments are needed to identify the chemical specie(s) of Roundup mixture involved in DNA adduct formation. Findings of this study may help to protect agricultural workers from health hazards and provide a basis for risk assessment. Environ. Mol. Mutagen. 31:55–59, 1998.

A retro-inverso peptide homologous to helix 1 of c-Myc is a potent and specific inhibitor of proliferation in different cellular systems

In 1998 we reported that an L‐peptide derived from H1 of c‐Myc (Int‐H1‐S6A,F8A), linked to an internalization sequence from the third α‐helix of Antennapedia, was endowed with an antiproliferative and proapoptotic activity toward a human mammary cancer cell line: The activity apparently depends upon the presence of the Myc motif. In the present work we have added new dimensions to our original findings. It is known that short retro‐inverso (RI‐) peptides can assume a 3D conformation very close to their corresponding L‐forms and can be recognized by the same monoclonal antibody. We synthesized a RI‐peptide form of our original L‐peptide: It was much more resistant to serum peptidases than the original molecule (a half life of days rather than hours); in addition, the RI‐form of the original Antennapedia internalization sequence was perfectly capable of carrying a D‐peptide into human cells. We have studied three different potentially active peptides. L‐peptides: Int‐H1wt, Int‐H1‐S6A,F8A. D‐peptides: RI‐Int‐H1‐S6A,F8A. We have also studied three presumed control peptides: Int and RI‐Int (no H1 motif), H1‐S6A,F8A (no internalization sequence). Both ‘active’ and ‘control’ peptides have essentially confirmed our expectations, however, in cells treated with the higher concentration (10 μM) of the control peptide RI‐Int, non‐Myc related side effects could be detected. In order to investigate whether the antiproliferative activities displayed by some of our molecules were indeed related to an interference with the role of c‐Myc (and molecules of the family), we chose an iso‐amphipathic modified peptide of the H1 motif, with a proximity coefficient >50% and where the major change was at position 7 (F→A). From a family of 73 H1 motifs belonging to (H1‐Loop‐H2) human sequences, the smallest evolutionary distance from our reference peptide was observed for the H1 of N‐Myc, L‐Myc, c‐Myc, H1‐S6A,F8A of c‐Myc, and Max, in that order. Our reference peptide was therefore appropriate as a check of whether we were indeed observing activities related to Myc functions. Both Int‐H1isoamph and the corresponding RI‐Int‐H1isoamph peptide were synthesized and studied. In terms of biological targets, we added to the human mammary cancer line of our previous work (MCF‐7 cells) a colon cancer line (HCT‐116 cells) and also a system of normal cells: human peripheral blood lymphocytes (PBLs) stimulated with phytohemoagglutinin (PHA). Peptides carrying an iso‐amphipathic‐modified H1 sequence were always very clearly (3‐10 times) less active than the corresponding peptides carrying a conserved “H1 of Myc” motif. This finding was noted in five independent situations (all the cellular models considered at the present time): MCF‐7 cells treated with L‐peptides; MCF‐7 cells treated with RI‐peptides; HCT‐116 cells treated with L‐peptides; PBLs treated with L‐peptides; PBLs treated with RI‐peptides. Modulation of transcription levels of ornithine decarboxylase (ODC), p53, and glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH), in PBLs treated with our different molecules, was well compatible with an interference by our active peptides at the level of Myc transcriptional activity. We had already reported a similar observation in MCF‐7 cells. On a molar basis, RI‐peptides were about 5–10 times more potent and 30–35 times more stable in complete culture medium, than their corresponding L‐forms. RI‐Int can probably internalize longer peptido‐mimetic molecules (for instance molecules mimetic of (H1‐Loop‐H2), or even more. These possibilities open the way to rodent studies and to more potent/selective Myc inhibitors—two steps closer to a potential drug.

DDP-induced cytotoxicity is not influenced by p53 in nine human ovarian cancer cell lines with different p53 status.

Nine human ovarian cancer cell lines that express wild-type (wt) or mutated (mut) p53 were used to evaluate the cytotoxicity induced by cisplatin (DDP). The concentrations inhibiting the growth by 50% (IC50) were calculated for each cell line, and no differences were found between cells expressing wt p53 and mut p53. Using, for each cell line, the DDP IC50, we found that these concentrations were able to induce an increase in p53 levels in all four wt-p53-expressing cell lines and in one out of five mut-p53-expressing cell lines. WAF1 and GADD45 mRNAs were also increased by DDP treatment, independently of the presence of a wt p53. Bax levels were only marginally affected by DDP, and this was observed in both wt-p53- and mut-p53-expressing cells. DDP-induced apoptosis was evident 72 h after treatment, and the percentage of cells undergoing apoptosis was slightly higher for wt-p53-expressing cells. However, at doses near the IC50, the percentage of apoptotic cells was less than 20% in all the cell lines investigated. We conclude that the presence of wt p53 is not a determinant for the cytotoxicity induced by DDP in human ovarian cancer cell lines.

An intranuclear frame for chromatin compartmentalization and higher-order folding

Recent ultrastructural, immunoelectron, and confocal microscopy observations done in our laboratory [Barboro et al. [2002] Exp. Cell. Res. 279:202–218] have confirmed that lamins and the nuclear mitotic apparatus protein (NuMA) are localized inside the interphase nucleus in a polymerized form. This provided evidence of the existence of a RNA stabilized lamin/NuMA frame, consisting of a web of thin (∼3 and ∼5 nm) lamin filaments to which NuMA is anchored mainly in the form of discrete islands, which might correspond to the minilattices described by Harborth et al. [1999] (EMBO. J. 18:1689–1700). In this article we propose that this scaffold is involved in the compartmentalization of both chromatin and functional domains and further determines the higher‐order nuclear organization. This hypothesis is strongly supported by the scrutiny of different structural transitions which occur inside the nucleus, such as chromatin displacement and rearrangements, the collapse of the internal nuclear matrix after RNA digestion and the disruption of chromosome territories induced by RNase A and high salt treatment. All of these destructive events directly depend on the loss of the stabilizing effect exerted on the different levels of structural organization by the interaction of RNA with lamins and/or NuMA. Therefore, the integrity of nuclear RNA must be safeguarded as far as possible to isolate the matrix in the native form. This material will allow for the first time the unambiguous ultrastructural localization inside the INM of the components of the functional domains, so opening new avenues of investigation on the mechanisms of gene expression in eukaryotes.

Molecular Interaction Maps--A Diagrammatic Graphical Language for Bioregulatory Networks

Molecular interaction maps (MIMs) use a clear, accurate, and versatile graphical language to depict complex biological processes. Here, we discuss the main features of the MIM language and its potential uses. MIMs can be used as database resources and simulation guides, and can serve to generate new hypotheses regarding the roles of specific molecules in the bioregulatory networks that control progression through the cell cycle, differentiation, and cell death.

Changes in the expression of cytokeratins and nuclear matrix proteins are correlated with the level of differentiation in human prostate cancer

The nuclear matrix‐intermediate filament complex (NM‐IF) is a protein scaffold which spans the whole cell, and several lines of evidence suggest that this structural frame represents also a functional unit, which could be involved in the epigenetic control of cancer development. Here we report the characterization by high resolution two‐dimensional gel electrophoresis and Western blot analysis of the NM‐IF complex isolated from prostate cancer (PCa); tumor‐associated proteins were identified by comparing the electrophoretic patterns with those of normal human prostate (NHP). Extensive changes in the expression of both the NM and IF proteins occur; they are, however, related in a different way to tumor progression. Poorly differentiated PCa (Gleason score 8–9) shows a strong down regulation of several constitutive cytokeratins (CKs 8, 18, and 19); their expression significantly (P < 0.05) decreases with respect to both NHP and benign prostatic hyperplasia (BPH) and, more interestingly, also with respect to moderately (Gleason score 6–7) and well (Gleason score 4–5) differentiated tumors. Moreover, we have identified a tumor‐associated species which is present in all of the tumors examined, systematically absent in NHP and occurs only in a few samples of BPH; this polypeptide, of Mr 48,000 and pI 6.0, represent a proteolytic fragment of CK8. At variance with these continuing alterations in the expression, the NM proteins undergo stepwise changes correlating with the level of differentiation. The development of less differentiated tumors is characterized by the appearance of several new proteins and by the decrease in the expression of others. Six proteins were found to be expressed with a frequency equal to one in poorly differentiated tumor, namely in all the samples of tumor examined, while in moderately and well differentiated tumors the frequency is less than one, and decreases with increasing the level of differentiation. When tumors of increasing Gleason score are compared with NHP a dramatic increase in the complexity of the protein patterns is observed, indicating that tumor dedifferentiation results in a considerable increase in the phenotypic diversity. These results suggest that tumor progression can be characterized using an appropriate subset of tumor‐associated NM proteins. J. Cell. Biochem. 79:471–485, 2000.

Potent synergistic interaction between the Nampt inhibitor APO866 and the apoptosis activator TRAIL in human leukemia cells

OBJECTIVE The nicotinamide phosphoribosyltransferase (Nampt) inhibitor APO866 depletes intracellular nicotinamide adenine dinucleotide (NAD(+)) and shows promising anticancer activity in preclinical studies. Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to plasma membrane receptors DR4 and DR5 and induces apoptosis via caspase-8 and -10. Here we have explored the interaction between APO866 and TRAIL in leukemia cell lines and in primary B-cell chronic lymphocytic leukemia cells. MATERIALS AND METHODS Cells were treated with APO866, TRAIL, or their combination. Viability and mitochondrial transmembrane potential (ΔΨ(m)) were determined by cell staining with propidium iodide and tetramethylrhodamine ethyl ester, respectively, and flow cytometry. Nampt and γ-tubulin levels, as well as caspase-3 cleavage were detected by immunoblotting. DR4 and DR5 expression were assessed by immunostaining and flow cytometry. Caspases were inhibited with zVAD-FMK and zDEVD-FMK; autophagy with 3-methyladenine, LY294002, and wortmannin. Intracellular NAD(+) and adenosine triphosphate (ATP) were measured by cycling assays and high-performance liquid chromatography (HPLC), respectively. RESULTS APO866 induced NAD(+) depletion, ΔΨ(m) dissipation, and ATP shortage in leukemia cells, thereby leading to autophagic cell death. TRAIL induced caspase-dependent apoptosis. TRAIL addition to APO866 synergistically increased its activity in leukemia cells by enhancing NAD(+) depletion, ΔΨ(m) dissipation, and ATP shortage. No DR5 upregulation at the cell surface in response to APO866 was observed. Remarkably, in healthy leukocytes APO866 and TRAIL were poorly active and failed to show any cooperation. CONCLUSIONS Activation of the extrinsic apoptotic cascade with TRAIL selectively amplifies the sequelae of Nampt inhibition in leukemia cells, and appears as a promising strategy to enhance APO866 activity in hematological malignancies.

Studies on DNA binding of caffeine and derivatives: evidence of intercalation by DNA-unwinding experiments

Caffeine and derivatives are compounds with pleiotropic effects on the genetic material which are supposed to originate from drugs binding to DNA. Here we show, by using two different topological methods, that methylated oxypurines, at biologically relevant concentrations, unwind DNA in a fashion similar to known intercalators. Methylated oxypurines could be ranked by decreasing unwinding potency: 8-methoxycaffeine greater than 8-ethoxycaffeine greater than 8-chlorocaffeine greater than caffeine greater than theophylline. These findings confirm, with a different assay, interaction of caffeine with DNA and add additional support to an intercalative mode of binding of these drugs to DNA.