Davide Malacarne

Azienda Ospedaliera Universitaria San Martino di Genova, Genova, Liguria, Italy

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Publications (33)159.16 Total impact

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    ABSTRACT: Objectives: Retrospective studies seem to support the role of DNA aneuploidy in oral leukoplakia (OL) as a risk factor for malignant transformation. Given the comparable risk of malignant transformation between non-dysplastic OL and oral lichen planus (OLP), similar DNA aneuploidy prevalence could be hypothesized. We assessed the DNA ploidy status in OL and OLP aiming to compare non-dysplastic homogeneous OL and reticular/papular OLP. Methods: Tissue samples were obtained from lesions on the buccal mucosa clinically suggestive for homogeneous OL or OLP with papular and reticular aspect and without atrophic- erosive aspect. Part of the sample was formalin-fixed to assess the presence of dysplasia or confirm the diagnosis of OLP; another part of the sample was frozen to determine the DNA content expressed by the DNA index (DI) by means of high-resolution DNA flow cytometry (hr DNA-FCM). A DI value different from 1 identified a DNA aneuploid status. The prevalence of DNA aneuploid status in OL and OLP was compared by Fisher’s exact test. Results: OL showed a DNA aneuploid status in 13.2% of lesions (5 out of 38), while two out of 44 samples from OLP lesions had a DNA aneuploid status (4.5%). The prevalence on aneuploidy was similar when comparing OL and OLP (P = 0.241; two-tailed Fisher’s exact test). Conclusion(s): Homogeneous non-dysplastic leukoplakia and white OLP from an oral subsite with low risk of malignant transformation have similar chromosomal instability as assessed by DNA ploidy. We are collecting further data to explore the potential role of a non-homogeneous clinical aspect in differentiating OL and OLP. Relevance: Non-dysplastic potentially malignant disorders from the same oral subsite and with similar clinical aspect have similar DNA aneuploidy prevalence. Oral Diseases Original Clinical Research 12
    12th Biennial Congress of the European Association of Oral Medicine, 11–14 September 2014, Gloria Hotels and Resort Antayla, Turkey, Supplement s2, pages 24–35; 09/2014
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    ABSTRACT: BACKGROUND: Chromosomal Instability and aneuploidy may represent biomarkers of oral exposure to damaging agents and early signs of clinical disease according to the theory of "oral field cancerization". METHODS: The hypothesis was tested that the DNA Index values, obtained by high-resolution DNA flow cytometry, may potentially contribute to oral cancer risk prediction. For this purpose, the DNA Index of oral fields of normal appearing mucosa and oral potentially malignant disorders in 165 consecutive patients was tested for association with dysplasia and/or the oral sub-sites of tongue and floor of the mouth taken as high-risk intermediate end-points surrogate of cancer clinical end-points. The association was evaluated by logistic regression using patient gender, age, tobacco cigarette smoking habit and alcohol abuse as confounding variables. RESULTS: Different DNA Index models provided evidence of statistical significant associations. Subdividing the DNA Index values in diploid, near-diploid aneuploid and high or multiple aneuploid from both oral potentially malignant disorders and oral normal appearing mucosa, Odds Ratios respectively of 1, 4.3 (p=0.001) and 18.4 (p<0.0005) were obtained. CONCLUSIONS: Routine DNA Index analysis by high-resolution DNA flow cytometry appears potentially useful to complement dysplasia and sub-site analysis for assessment of oral cancer risk-prediction and for a better management of the patients with oral potentially malignant disorders. Work is in progress to validate the present findings in a prospective study with clinical end-points. Impact: Identifying DNA abnormalities in oral pre-malignancy may lead to biomarkers of oral exposure and cancer risk and potentially to more effective prevention measures.
    Cancer Epidemiology Biomarkers & Prevention 04/2013; 22(6). DOI:10.1158/1055-9965.EPI-13-0147 · 4.32 Impact Factor
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    Cell cycle (Georgetown, Tex.) 09/2012; 11(19):3703. DOI:10.4161/cc.22288 · 5.01 Impact Factor
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    ABSTRACT: Oral fields of visually normal and non-dysplastic mucosa (ODFs) may represent the precursors of oral potentially malignant lesions (OPMLs). Aim of the study was to provide new evidence for the concept of the "field carcinogenesis" model by comparing the ODF and OPML genomic aberration profiles obtained by high resolution DNA flow cytometry (hr DNA-FCM) and array-Comparative Genomic Hybridization (a-CGH). A second aim was to investigate if specific CGH aberrations were associated with DNA aneuploidy. Nineteen patients with single OPMLs were recruited for the study. In parallel with obtaining samples of OPML tissue from 11 leukoplakias without dysplasia (nd-OPMLs) and 8 with dysplasia (d-OPMLs), we also obtained samples from distant ODFs. DNA aneuploid nuclei detected by hr DNA-FCM were physically separated, based on DNA content, from the DNA diploid components with a DNA-FCM-Sorter. These relatively pure subpopulations of epithelial nuclei were then submitted to DNA extraction and a-CGH for a genome-wide analysis of DNA copy number aberrations (CNAs). The frequencies of DNA aneuploidy (DI ≠ 1) among ODFs and OPMLs were respectively 5.3% and 32%. The DI aneuploid values of ODFs and nd-OPMLs were all near-diploid (DI ≠ 1 and DI ≤ 1.4), while for d-OPMLs were high-aneuploid (DI > 1.4) in 40% of the cases. CNA averages were 1.9 in ODFs and 6.5 in OPMLs. The gain of the chromosomal region 20q13.33-qter was observed in 37% of both ODFs and corresponding OPMLs. Additional common regions included 7p22.2-pter, 11p15.5-pter and 16p13.3-pter where gains were observed. Furthermore, gains of 20q13.31-q13.33 and of 5p13.33-pter and loss of 9p21.3 were detected at high frequency (respectively, at 62.5%, 50% and 50%) only in d-OPMLs. In particular, loss at 9p21.3, gain at 5p13.33-pter and gain of 20q13.31-q13.33 were associated with DNA aneuploidy (p = 0.00004; p = 0.0005; p = 0.01). ODFs and OPMLs showed common CNAs in specific chromosomal regions suggesting that they may represent early events of the natural history of oral carcinogenesis according to the field effect cancerization and may contribute to the ODF-OPML transition. In addition, loss at 9p21.3 and gains at 5p13.33-pter and 20q13.31-q13.33 may contribute to DNA aneuploidization.
    12/2011; 35(1):43-52. DOI:10.1007/s13402-011-0064-2
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    ABSTRACT: The mucosae of the oral cavity are different at the histological level but appear all equally exposed to common genotoxic agents. As a result of this exposure, changes in the mucosal epithelia may develop giving rise to Oral Potentially Malignant Lesions (OPMLs), which with time may in turn progress to Oral Squamous Cell Carcinomas (OSCCs). Therefore, much effort should be devoted to identify features able to predict the likeliness of progression associated with an OPML. Such features may be helpful in assisting the clinician to establish both appropriate therapies and follow-up schedules. Here, we report a pilot study that compared the occurrence of DNA aneuploidy and chromosomal copy number aberrations (CNAs) in the OPMLs from different oral anatomical subsites. Samples from histologically diagnosed OPMLs were processed for high resolution DNA flow cytometry (hr DNA-FCM) in order to determine the relative DNA content expressed by the DNA index (DI). Additionally, array-Comparative Genomic Hybridization (a-CGH) analysis was performed on DNA obtained from diploid nuclei suspensions directly. When aneuploid nuclei were detected, these were physically separated from diploid nuclei on the base of their DI values by means of a DNA-FCM-Sorter in order to improve the a-CGH analysis. Tongue OPMLs were more frequently associated with DNA aneuploidy and CNAs than OPMLs arising from all the other mucosal subsites. We suggest that the follow-up and the management of the patients with tongue OPMLs should receive a distinctive special attention. Clearly, this hypothesis should be validated in a prospective clinical study.
    BMC Cancer 10/2011; 11:445. DOI:10.1186/1471-2407-11-445 · 3.32 Impact Factor
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    ABSTRACT: To test the hypothesis that cigarette smokers develop oral potentially malignant disorders or carcinomas in preferential anatomical subsites. The association of smoking habit with the presence of oral lesions in specific anatomical subsites was assessed in 123 patients using the odds ratio analysis. When compared to all the other subsites, the relative frequency of smokers with lesions was higher in the buccal mucosa and in the floor of the mouth (FOM) (P=0.002 and P=0.005), while it was lower in the tongue (P<0.0005). Smokers were about 7 years younger than non-smokers (P=0.008). The association of smoking and age suggests that smoking may contribute to generate a field of injury that leads to lesions in shorter periods than other causes. The stronger relationship of smoking with lesions in the buccal mucosa and FOM than in the tongue suggests that tissue characteristics mediate the effects of tobacco.
    Journal of Oral Pathology and Medicine 03/2011; 40(3):214-7. DOI:10.1111/j.1600-0714.2010.00984.x · 1.87 Impact Factor
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    ABSTRACT: Oral potentially malignant lesions (OPMLs) with dysplasia and aneuploidy are thought to have a high risk of progression into oral squamous cell carcinomas (OSCCs). Non-dysplastic "oral distant fields" (ODFs), characterized by clinically normal appearing mucosa sited at a distance from co-existing OPMLs, and non-dysplastic OPMLs may also represent an early pre-cancerous state. ODFs, OPMLs without and with dysplasia and OSCCs were investigated by high resolution DNA content flow cytometry (FCM). ODFs and OPMLs without dysplasia were DNA aneuploid respectively in 7/82 (8.5%) and 25/109 (23%) cases. "True normal oral mucosa" and human lymphocytes from healthy donors were DNA diploid in all cases and were used as sex specific DNA diploid controls. Dysplastic OPMLs and OSCCs were DNA aneuploid in 12/26 (46%) and 12/13 (92%) cases. The DNA aneuploid sublines were characterized by the DNA Index (DI not =1). Aneuploid sublines in ODFs and in non-dysplastic and dysplastic OPMLs were near-diploid (DI<1.4) respectively in all, 2/3 and 1/3 of the cases. DNA aneuploid OSCCs, instead, were characterized prevalently by multiple aneuploid sublines (67%), which were commonly (57%) high-aneuploid (DI> or =1.4). DNA near-diploid aneuploid sublines in ODFs and OPMLs appear as early events of the oral carcinogenesis in agreement with the concept of field effect. Near-diploid aneuploidization is likely to reflect mechanisms of loss of symmetry in the chromosome mitotic division. High DNA aneuploid and multiple sublines in OPMLs with dysplasia and OSCCs suggest, instead, mechanisms of "endoreduplication" of diploid and near-diploid aneuploid cells and chromosomal loss. High resolution DNA FCM seems to enable the separation of subsequent progression steps of the oral carcinogenesis.
    Cellular oncology: the official journal of the International Society for Cellular Oncology 01/2010; 32(5-6):373-83. DOI:10.3233/CLO-2010-0525 · 4.17 Impact Factor
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    ABSTRACT: To date there are still no reliable biomarkers for oral potentially malignant disorders (PMDs) to predict the risk of progression to squamous cell carcinoma (SCC). Within a prospective clinical trial of patients with PMDs, DNA content flow cytometry (DNA FCM) was evaluated for 60 PMDs using fresh samples obtained by a dermatological curette. There were 6/42 PMDs without dysplasia, but with DNA aneuploidy, versus 8/18 with both dysplasia and aneuploidy (p=0.02). When the tongue and the buccal mucosa, the two most common sites in the present series of cases were compared, dysplastic PMDs were mainly located on the tongue (p=0.01). Tobacco smokers, who preferentially developed PMDs in the buccal mucosa at a younger age than non-smokers (p=0.002), had fewer dysplastic PMDs than did non-smokers (p=0.01). Dysplasia was significantly linked to DNA aneuploidy (p=0.03) in smokers. The present data suggest that aneuploidy is an early event in oral carcinogenesis and that the influence of tobacco varies according to subsite and patient age. When DNA FCM of PMD samples are obtained by curette scraping, extensive areas can be covered with a minimally invasive, rapid, inexpensive procedure. Moreover DNA FCM of these samples appears easy amenable to routine analysis. Further research on larger numbers of PMDs should be carried out to determine whether DNA FCM plays a role in the prediction of risk of PMD transformation.
    Oral Oncology 06/2009; 45(10):887-90. DOI:10.1016/j.oraloncology.2009.03.008 · 3.03 Impact Factor
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    ABSTRACT: BH3-only members of the Bcl-2 family exert a fundamental role in apoptosis induction. This work focuses on the development of a novel peptidic molecule based on the BH3 domain of Bim. The antiapoptotic molecule Bcl-X(L), involved in cancer development/progression and tumour resistance to cytotoxic drugs, is a target for Bim. According to a rational study of the structural interactions between wt Bim-BH3 and Bcl-X(L), we replaced specific residues of Bim-BH3 with natural and non-natural aminoacids and added an internalizing sequence, thus increasing dramatically the inhibitory activity of our modified Bim-BH3 peptide, called 072RB. Confocal microscopy and flow cytometry demonstrated cellular uptake and internalization of 072RB, followed by co-localization with mitochondria. Multiparameter flow cytometry demonstrated that the 072RB dose-dependent growth inhibition of leukaemia cell lines was due to apoptotic cell death. No effect was observed when cells were treated with the internalizing vector alone or a mutated control peptide (single aminoacid substitution L94A). Ex-vivo derived leukemic cells from acute myeloid leukaemia (AML) patients underwent cell death when cultured in vitro in the presence of 072RB. Conversely, no significant cytotoxic effect was observed when 072RB was administered to cultures of peripheral blood mononuclear cells, either resting or PHA-stimulated, and bone marrow cells of normal donors. Xenografts of human AML cells in NOD/SCID mice displayed a significant delay of leukemic cell growth upon treatment with 072RB administered intravenously (15 mg/Kg three times, 48 hours after tumour cell injection). Altogether, these observations support the therapeutic potentials of this novel BH3 mimetic.
    Cell cycle (Georgetown, Tex.) 11/2008; 7(20):3211-24. DOI:10.4161/cc.7.20.6830 · 5.01 Impact Factor
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    ABSTRACT: c-Myc is a transcription modulator proto-oncogene. When overexpressed, it becomes an important contributor to the multi-hit process of malignant transformation. In two earlier papers in this journal (see refs. 19 , 20) we reported that retro-inverso peptidomimetic molecules inspired by the Helix-1 of c-Myc motif could be sequence-specific antiproliferative agents active in the low micromolar range. We also found that our peptides were not opening the four-alpha-helix Myc:Max bundle. Their antiproliferative activity in cancer cell lines needs the presence of side chains projecting outside of the bundle in the corresponding native H1 motif. This observation suggested interference with an external partner. In this study we investigated the INI1:Myc interaction. INI1 is a subunit of the SWI/SNF complex (component of the enhanceosome surrounding Myc:Max heterodimer). The INI1:Myc interaction was confirmed via pull down, ELISA, and fluorescence anisotropy assays. According to the length of INI1 fragments used, we calculated Kds ranging between 1.3x10(-6) and 4.8x10(-7) M. The three different techniques applied showed that the INI1:Myc interaction was also the target of our retro-inverso peptidomimetic molecules, which seem to bind specifically at INI1. A Myc binding, 21aa INI1 fragment (minimum interacting sequence), could inspire the synthesis of a new class of more selective c-Myc inhibitors.
    The FASEB Journal 05/2007; 21(4):1256-63. DOI:10.1096/fj.06-7082com · 5.48 Impact Factor
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    ABSTRACT: The origin and mechanisms of chromosomal instability are still widely unknown. We previously investigated a limited number of human sporadic colorectal cancers (CRCs) and observed a statistically different occurrence of KRAS and p53 mutations among predetermined subgroups of tumors with different degrees of DNA aneuploidy. The aim of the present study was to further verify these observations by including BRAF gene analysis and by investigating a larger series of cases subdivided into Dukes' stages A to D to reconstruct some form of chronological modulation for events during CRC progression. KRAS, p53, BRAF mutations and flow cytometric DNA Index were evaluated by established techniques in a series of 135 human sporadic CRCs. p53, KRAS and BRAF mutations were found in 39%, 34%, and 4% of tumors, respectively. The frequency of p53 mutations increased from 15% for stage A to 48% for stage D and was highest in near-diploid (DI < 1.4 and DI does not equal 1) and high-aneuploid (DI > 1.6) tumors. A similar correlation between gene mutations and DI values was observed for KRAS. The simultaneous presence of KRAS and p53 mutations was observed in only 11% of cases. Moreover, the co-occurrence of p53 and KRAS mutations was only observed in near-diploid and high-aneuploid tumors. Our findings suggest that KRAS and p53 gene mutations, which are rarely simultaneous and are associated with specific DI aneuploid values, do not represent a synergistic evolutionary pathway but may influence mechanisms of chromosomal instability.
    Cellular oncology: the official journal of the International Society for Cellular Oncology 01/2006; 28(4):161-6. · 4.17 Impact Factor
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    ABSTRACT: Despite the best and most aggressive, often integrated, standard therapeutic approaches for mesothelioma, overall survival remains very poor. The actual failure points out clearly the need for the development of novel therapy. One of the promising paths of experimentation is artificial induction of apoptosis. A therapeutic strategy that relies on the down-regulation of BCL-XL inhibition nuclear factor kappaB (NF-kappaB) with a combination of SN38 and tumor necrosis factor (TNF) was studied in human mesothelioma cell lines (MSTO-221H, IST-MES1, IST-MES2, MPP89, H28, H513, H2052, and H290). Cell proliferation (clonogenic assay) was inhibited strongly by the combination of TNF and SN38. Examining the persistence of the NF-kappaB complexes using an electrophoretic mobility-shift assay, it appeared that they still were present at 24 hours in TNF-treated cells. In SN38-treated cells, NF-kappaB complexes persisted for 6 hours. In cells that were treated with combined SN38 and TNF, NF-kappaB complexes disappeared quickly and became undetectable at 6 hours. In flow cytometry analysis, only cells that were treated with combined SN38 and TNF demonstrated significant cellular accumulation in the sub-G0-G1 phase, suggesting a specific induction of apoptosis. Morphologic examination (4,6-diamidino-2-phenylindole staining and electron microscopy) and internucleosomal DNA fragmentation (gel ladder) confirmed rigorously the induction of apoptosis. Because of NF-kappaB inhibition with the combination of SN38 and TNF, the expression of BCL-XL (both the protein [Western blot analysis] and the mRNA [reverse transcriptase-polymerase chain reaction analysis]) was down-regulated, cytochrome c was released into the cytoplasm, caspase 3 was activated (Western blot analysis), and, consequently, apoptosis was triggered. The authors hope that the results of the current study may contribute to the design and implementation of a novel therapeutic approach that improves patients' responses to treatment for mesothelioma.
    Cancer 05/2005; 103(7):1503-18. · 4.90 Impact Factor
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    ABSTRACT: Our work is focused in the broad area of strategies and efforts to inhibit protein-protein interactions. The possible strategies in this field are definitely much more varied than in the case of ATP-pocket inhibitors. In our previous work (10), we reported that a retro-inverso (RI) form of Helix1 (H1) of c-Myc, linked to an RI-internalization sequence arising from the third alpha-helix of Antennapedia (Int) was endowed with an antiproliferative and proapoptotic activity toward the cancer cell lines MCF-7 and HCT-116. The activity apparently was dependent upon the presence of the Myc motif. In this work, by ala-scan mapping of the H1 portion of our molecules with D-aa, we found two amino acids necessary for antiproliferative activity: D-Lys in 4 and D-Arg in 5 (numbers refer to L-forms). In the natural hetero-dimer, these two side chains project to the outside of the four alpha-helix bundle. Moreover, we were able to obtain three peptides more active than the original lead. They strongly reduced cell proliferation and survival (RI-Int-VV-H1-E2A,S6A,F8A; RI-Int-VV-H1-S6A,F8A,R11A; RI-Int-VV-H1-S6A,F8A,Q13A): after 8 days at 10 muM total cell number was approximately 1% of the number of cells initially seeded. In these more potent molecules, the ablated side chains project to the inside in the corresponding natural four alpha-helix bundle. In the present work, we also investigated the behavior of our molecules at the biochemical level. Using both a circular dichroism (CD) and a fluorescence anisotropy approach, we noted that side chains projecting at the interior of the four alpha-helix bundle are needed for inducing the partial unfolding of Myc-H2, without an opening of the leucine zipper. Side chains projecting at the outside are not required for this biochemical effect. However, antiproliferative activity had the opposite requirements: side chains projecting at the outside of the bundle were essential, and, on the contrary, ablation of one side chain at a time projecting at the inside increased rather than decreased biological activity. We conclude that our active molecules probably interfere at the level of a protein-protein interaction between Myc-Max and a third protein of the transcription complex. Finally, CD and nuclear magnetic resonance (NMR) data, plus dynamic simulations, suggest a prevalent random coil conformation of the H1 portion of our molecules, at least in diluted solutions. The introduction of a kink (substitution with proline in positions 5 or 7) led to an important reduction of biological activity. We have also synthesized a longer peptido-mimetic molecule (RI-Int-H1-S6A,F8A-loop-H2) with the intent of obtaining a wider zone of interaction and a stronger interference at the level of the higher-order structure (enhanceosome). RI-Int-H1-S6A,F8A-loop-H2 was less active rather than more active in respect to RI-Int-VV-H1-S6A,F8A, apparently because it has a clear bent to form a beta-sheet (CD and NMR data).
    The FASEB Journal 05/2005; 19(6):632-4. DOI:10.1096/fj.04-2369fje · 5.48 Impact Factor
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    ABSTRACT: In the April 1, 2005, issue of Cancer, an article entitled “Tumor necrosis factor enhances SN38-mediated apoptosis in mesothelioma cells: The role of nuclear factor-κB pathway activation” was published by Dr. Russo and colleagues. On October 6, 2009, we were alerted to concerns about the integrity of the data in the article. A formal investigation was conducted by the Institutional Office for Research Integrity (UIR) at the National Institute for Cancer Research (IST) in Genoa, Italy. The investigation report from the UIR President, dated November 4, 2009, stated the following:“1) The internal investigation is concluded and no further analyses are planned on the issue.2) The UIR concluded that the article contains evidence of data fabrication/duplication.3) All the authors (either belonging or not to the IST) have been informed of the concerns about data integrity. Some authors acknowledged the conclusion stated in the previous point 2, others did not; however, none of them accepted the responsibility of having fabricated the data.”Based on this report and our concerns about the validity of the study, we hereby retract this article from Cancer and from the medical literature.Raphael E. Pollock, MD, PhD
    Cancer 04/2005; 103(7). DOI:10.1002/cncr.20924 · 4.90 Impact Factor
  • Mauro Risio, Davide Malacarne, Walter Giaretti
    Cellular oncology: the official journal of the International Society for Cellular Oncology 02/2005; 27(5-6):363-6. · 4.17 Impact Factor
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    ABSTRACT: The role of p53 in modifying sensitivity to cytotoxic drugs has been commonly studied by creating transfection pairs of wt p53 parental cells and altered p53 daughter cells, or vice versa. Authors inevitably tended to extrapolate and generalize their experimental observations, and conflicting reports have been more the rule than the exception. We have performed a meta-analysis of 356 independent studies. Average changes of drug sensitivity after a change of p53 status were observed. E6 transfection predominantly induces sensitization to cytotoxic drugs, whereas p53-/- knockout cells are more drug-resistant than their normal p53+/+ counterpart. Unexpectedly, transfection with a mutated p53 does not change much the drug sensitivity of most wt p53 cancer lines, with the notable exception of A2780, a predominant cell line in the studies analyzed (A2780 cells show increased resistance after transfection with a mutated p53). Rather interestingly, mitotic spindle poisons acted differently from other classes of cytotoxic drugs. A crucial indication of our findings is that the role of p53 alone in determining sensitivity/resistance to cytotoxic drugs is limited: the individual molecular pathology and differentiation of a given cancer line prevail over any average trend, and are causal to a broad spreading of the data. We also identify major "confounding factors", alias independent categorical variables, capable of affecting the average outcome.
    Biochimica et Biophysica Acta 01/2005; 1705(2):103-20. DOI:10.1016/j.bbcan.2004.10.001 · 4.66 Impact Factor
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    ABSTRACT: A new non peptidic farnesyltransferase inhibitor, RPR-115135, in combination with 5-FU was studied in 10 human colon cancer cell lines (HCT-116, RKO, DLD-1, Colo-320, LoVo, SW-620, HT-29, HCT-15, Colo-205 and KM-12) carrying several mutations but well characterized for p53 and Ras status. We found that there was a slight tendency (not statistically significant) for the p53 inactivated cells to be less sensitive to 5-FU after 6 days continuous treatment. Simultaneous administration of RPR-115135 and 5-FU, at subtoxic concentrations, resulted in a synergistic enhancement of 5-FU cytotoxicity in the p53 wildtype cells (HCT-116, RKO, DLD-1, Colo-320, LoVo). In the p53 mutated cells (SW-620, HT-29, HCT-15, Colo-205, KM-12) the effect was very complicated. In HCT-15 the combination resulted in antagonism, in KM-12 in antagonism or in synergy (at different concentrations) and in SW-620, HT-29 and Colo-205 cells in synergy but only when 5-FU was administered at high concentrations. Growth inhibition could be accounted for on the basis of a specific cell cycle arrest phenotype (G2-M arrest), as assayed by flow cytometry, only in the p53 functioning cell lines. The combination RPR-115135 + 5-FU increases apoptotic events only in these cell lines. In the mutated cell lines no major alterations on cell cycle arrest phenotype and no induction of apoptosis was observed. Although RPR-115135 can potentiate the effect of 5-FU in cells in which p53 function is disrupted, these data suggest strongly that RPR-115135 significantly enhances the efficacy of 5-FU only when p53 is functioning.
    International Journal of Cancer 07/2002; 100(3):266-75. DOI:10.1002/ijc.10461 · 5.01 Impact Factor
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    ABSTRACT: A new nonpeptidic farnesyltransferase inhibitor, RPR-115135, in combination with 5-fluorouracil (5-FU) was studied in an isogenic cell line model system consisting of human colon cancer HCT-116 cells. HCT-116 cells were transfected with an empty control pCMV vector and with a dominant-negative mutated p53 transgene (248R/W). We found that, relative to control transfectants, there was a slight tendency for the p53 inactivated cells to be less sensitive to 5-FU after 6 days of continuous treatment. Simultaneous administration of RPR-115135 and 5-FU, at equitoxic concentrations, resulted in an enhancement of 5-FU cytotoxicity, especially in the CMV-2 clone. Growth inhibition could be accounted for on the basis of a specific cell cycle arrest phenotype (G(2)-M arrest in CMV-2 and S arrest in mutated clones), as assayed by flow cytometry. The combination RPR-115135 + 5-FU increases apoptotic events only in the CMV-2 clone.
    Journal of Pharmacology and Experimental Therapeutics 02/2002; 300(1):220-6. · 3.86 Impact Factor
  • Nature Genetics 04/2001; 27(4):77-78. DOI:10.1038/87238 · 29.65 Impact Factor
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    ABSTRACT: 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 a-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 mM) 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) hu man 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.
    The FASEB Journal 02/2001; 15(1):31-33. DOI:10.1096/fj.00-0422fje · 5.48 Impact Factor

Publication Stats

420 Citations
159.16 Total Impact Points

Institutions

  • 2013
    • Azienda Ospedaliera Universitaria San Martino di Genova
      Genova, Liguria, Italy
  • 2008–2012
    • CRO Centro di Riferimento Oncologico di Aviano
      • Division of Experimental Oncology 1
      Aviano, Friuli Venezia Giulia, Italy
  • 1994–2007
    • Università degli Studi di Genova
      • Dipartimento di Medicina sperimentale (DIMES)
      Genova, Liguria, Italy
  • 2006
    • Azienda Unità Sanitaria Locale Forlì
      Forlì, Emilia-Romagna, Italy