How much do GOLD stages reflect CT abnormalities in COPD patients?

PURPOSE: Severity of chronic obstructive pulmonary disease (COPD) can be graded using the classification released in the Global Initiative for Chronic Obstructive Lung Disease (GOLD) report. Such classification is essentially based on spirometry and does not recognise the role of other measures. The aim of this study was to assess whether the GOLD stages correlate with the extent of pulmonary emphysema and other ancillary computed tomography CT features in a population of smokers with stable COPD. MATERIALS AND METHODS: Based on clinical assessment and lung-function testing, patients were classified according to the GOLD criteria. CT scans were visually evaluated for extent of emphysema and airway abnormalities. RESULTS: A total of 43 patients were enrolled. The amount of emphysema was described as minimal in six patients with stage 0, and as moderate in seven patients with stage 0. In stages I and II, the extent of emphysema ranged from minimal to severe, whereas we observed the presence of severe emphysema in most patients in stages III and IV. According to the regression model, only CT emphysema extent independently predicted the GOLD stage (r2 = 0.58; p < 0.001). The cutoff value of emphysema extent of 31.5% allowed us to distinguish patients with a GOLD stage > or = III. CONCLUSIONS: Although we found a significant correlation between CT emphysema extent and GOLD stages, different percentage of emphysema extent can be observed among each GOLD stage. The upper limit of 31.5% of emphysema extent may indicate a boundary for a clinically worsening status.

Sequence specific peptidomimetic molecules inhibitors of a protein-protein interaction at the helix 1 level of c-Myc.

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).

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 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.

Inhibition of cancer cell growth and c-Myc transcriptional activity by a c-Myc helix 1-type peptide fused to an internalization sequence.

c-Myc is a nuclear protein with important roles in cell transformation, cell proliferation, and gene transcription. It has been previously shown that a 14-amino acid (aa) modified peptide (H1-S6A,F8A) derived from the helix 1 (H1) carboxylic region of c-Myc can interfere in vitro with specific c-Myc DNA binding. Here, we have linked the above Myc-derived 14-aa peptide to a 16-aa sequence from the third helix of Antennapedia (Int). It has been repeatedly reported that this 16-aa Antennapedia peptide is able to cross mammalian cell membranes and to work as a vector for short peptides. Using fluorescent (dansylated or rhodaminated) peptides, we have shown that the fusion peptide with the Antennapedia fragment (Int-H1-S6A,F8A) but not the c-Myc derived fragment alone (H1-S6A,F8A) was capable of internalization inside MCF-7 human breast cancer cells. Int-H1-S6A,F8A and H1-S6A,F8A were the only two peptides capable of inhibiting coimmunoprecipitation of the c-Myc/Max heterodimer in vitro. We have treated (continuously for 10-11 days) MCF-7 cells with four different peptides: Int, H1-S6A,F8A, Int-H1-S6A,F8A, and Int-H1wt [a peptide differing from Int-H1-S6A,F8A by 2 aa (S6 and F8) in the H1 region]. In intact MCF-7 cells, Int-H1-S6A,F8A was the only active peptide capable of inducing the following biological effects: (a) inhibition of cloning efficiency on plates; (b) inhibition of cell growth and induction of apoptosis in subconfluent/confluent cells; and (c) inhibition of transcription of two c-Myc-regulated genes (ODC and p53). Int-H1-S6A,F8A was active in the 1-10 microM range. Int-H1-S6A,F8A may represent a lead molecule for peptidomimetic compounds that have a similar three-dimensional structure but are more resistant to peptidases and, therefore, suitable for in vivo treatment of experimentally induced tumors.

Mutation frequencies at codon 248 of the p53 tumour suppressor gene are not increased in colon cancer cell lines with the RER+ phenotype.

The replication-error positive (RER+) phenotype characterizes tumour cells with microsatellite instability. This 'mutator phenotype' is thought to induce spread mutations throughout the genome, thus increasing the risk of tumour development. Here we analyse spontaneously arising mutations at the tetranucleotide CCGG ( Msp I recognition site), at positions 14 067-14 070 of the p53 gene sequence, in three colon cancer cell lines, two with microsatellite instability and one without this characteristic. This restriction site covers hot-spot codon 248, which is often mutated in colon carcinomas. Using the Msp I RFLP-PCR assay we found that the mean mutation frequency at this site was not different among the cell lines considered. Taking the substitutions separately, none of the mutations involving codon 248 arose with significantly higher frequency in each of the RER+ cell lines (HCT116 and DLD1) compared with the RER-one (SW480). Only the CG transversion at nt 14 067 (codon 247) occurred with a slightly higher, but biologically insignificant, frequency in one of the RER+ cell lines (HCT116). Our in vitro data support the previously reported lack of correlation between microsatellite instability and p53 mutations in RER+ tumour specimens.

Methods for predicting carcinogenic hazards: new opportunities coming from recent developments in molecular oncology and SAR studies.

Without epidemiological evidence, and prior to either short-term tests of genotoxicity or long-term tests of carcinogenicity in rodents, an initial level of information about the carcinogenic hazard of a chemical that perhaps has been designed on paper, but never synthesized, can be provided by structure-activity relationship (SAR) studies. Herein, we have reviewed the interesting strategies developed by human experts and/or computerized approaches for the identification of structural alerts that can denote the possible presence of a carcinogenic hazard in a novel molecule. At a higher level of information, immediately below epidemiological evidence, we have discussed carcinogenicity experiments performed in new types of genetically engineered small rodents. If a dominant oncogene is already mutated, or if an allele of a recessive oncogene is inactivated, we have a model animal with (n-1) stages in the process of carcinogenesis. Both genotoxic and receptor-mediated carcinogens can induce cancers in 20-40% of the time required for classical murine strains. We have described the first interesting results obtained using these new artificial animal models for carcinogenicity studies. We have also briefly discussed other types of engineered mice (lac operon transgenic mice) that are especially suitable for detecting mutagenic effects in a broad spectrum of organs and tissues and that can help to establish mechanistic correlations between mutations and cancer frequencies in specific target organs. Finally, we have reviewed two complementary methods that, while obviously also feasible in rodents, are especially suitable for biomonitoring studies. We have illustrated some of the advantages and drawbacks related to the detection of DNA adducts in target and surrogate tissues using the 32P-DNA postlabeling technique, and we have discussed the possibility of biomonitoring mutations in different human target organs using a molecular technique that combines the activity of restriction enzymes with polymerase chain reaction (RFLP/PCR). Prediction of carcinogenic hazard and biomonitoring are very wide-ranging areas of investigation. We have therefore selected five different subfields for which we felt that interesting innovations have been introduced in the last few years. We have made no attempt to systematically cover the entire area: such an endeavor would have produced a book instead of a review article.

A fast and simple in situ PCR method to detect human papilloma virus infection in archival paraffin-embedded tissues.

We describe a simple and fast method for the detection and localization of low copy numbers of HPV DNA in formalin-fixed and paraffin-embedded archival tissues. We have developed a protocol for direct bl situ-PCR in order to demonstrate its convenience in rapid and reproducible assessment of HPV infection in unknown biopsies. The morphological aspect of the tissues has been maintained, despite the multiple steps of fixation, permeabilization and thermal cycling, and positivity has been detected only in virus target cells.