Internalization via Antennapedia protein transduction domain of an scFv antibody toward c-Myc protein.

We constructed a single-chain variable fragment miniantibody (G11-scFv) directed toward the transactivation domain of c-Myc, which is fused with the internalization domain Int of Antennapedia at its carboxyl terminus (a cargo-carrier construct). In ELISA experiments, an EC(50) for binding saturation was achieved at concentrations of G11-scFv-Int(-) of approximately 10(-8) M. Internalization of a fluoresceinated Fl-G11-scFv-Int(+) construct was observed in intact human cultured cells with confocal microscopy. After 5 h of incubation in medium containing 1 microM Fl-G11-scFv-Int(+) or Fl-G11-scFv-Int(-), fluorescence intensity was determined in individual cells, both for cytoplasmic and nuclear compartments: concentration levels of Fl-G11-scFv-Int(+), relative to the extracellular culture medium concentration, were 4-5 times higher in the cytoplasm, 7-8 times higher in the nucleus, and 10 times higher in the nucleoli. In the same experimental conditions, the Fl-G11-scFv-Int(-) construct was 3-4 times more concentrated outside of the cells than inside. Cell membranes kept their integrity after 5 h of incubation. The antiproliferative activity of our miniantibody was studied on HCT116 cells. Incubation with 4 microM G11-scFv-Int(+) for 4 days induced very significant statistical and biological growth inhibition, whereas Int alone was completely inactive. Miniantibodies capable of penetrating cell membranes dramatically broaden the potential for innovative therapeutic agents and attack of new targets.

Inhibition of a protein-protein interaction between INI1 and c-Myc by small peptidomimetic molecules inspired by Helix-1 of c-Myc: identification of a new target of potential antineoplastic interest.

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.

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).

Influence of age, sex, and growth variables on phalangeal quantitative ultrasound measures: a study in healthy children and adolescents.

Skeletal status by phalangeal quantitative osteosonography (DBM Sonic BP-IGEA) was examined in 1227 healthy children (641 boys and 586 girls) aged 3-16 years. Aims of the study were to evaluate some physical parameters pertaining to the ultrasound transmission crossing the phalanx in a school-age population and to relate these values to age, sex, and growth variables. A correlation was found between AD-SoS (amplitude-dependent speed of sound) and BTT (bone transmission time) and, age, height, weight, and pubertal stage, respectively. No correlation existed between FWA (fast wave amplitude) and SDy (dynamics of the ultrasound signal) and age, height, weight, pubertal stage, and BMI, respectively. AD-SoS increased in boys until 7-8 years of age. Thereafter a plateau was reached up to age 12-13 years, when a rapid increase was observed corresponding to pubertal growth rate acceleration. In girls, AD-SoS increased with age up to 10-11 years with a steeper increase at the time of puberty starting about 2 years earlier than in boys. BTT presented a similar trend. Mean AD-SoS values increased from Tanner pubertal stages 1 to 2 and from stage 3 to 4 in both sexes. Significantly higher mean AD-SoS values in stages 2, 3, and 4 were observed in girls as compared to boys. Mean BTT values increased significantly from stage 1 to 5 in girls and from 1 to 4 in boys. QUS technology showed the ability to assess bone changes in the growing bone.

162 HIV-1 infected pregnant women and vertical transmission. Results of a prospective study.

BACKGROUND: Aim of this paper is to describe the changes over a 16-year period of the characteristics and management of HIV infected pregnant women. METHODS: Prospective study: analysis of data obtained from 162 women and 176 infants. Factors evaluated included: maternal socio-demographic level, immunological and virological parameters, antiretroviral therapy, mode of delivery, pregnancy outcome and babies follow-up. RESULTS: The proportion of women with heterosexual acquisition of infection has increased significantly from 13.5% in 1985-1989 to 47.1% in 1996-2001 (p<0.0005, Fisher's exact test), while the proportion acquiring HIV through injecting drugs has declined. Mean CD4 cell count at delivery was 535 x 106/l (+/-522.3 x 106/l). In 1990, 50% of mothers received antiretroviral therapy, rising significantly to 87.5% in 2000. The elective cesarean section was introduced in 1998 and its rate has increased to 75% in 2000. The vertical transmission rate changed from 9.5% in 1985-1989 to 14.3% in 1996-2000 (this difference was not statistically significant, Fisher's exact test). CONCLUSIONS: Social characteristics of the HIV-infected women have changed since the mid-1980s: in recent times women are having children at increasingly older ages and are more likely to know that they are HIV infected when they become pregnant. Antiretroviral therapy, elective caesarean delivery and avoidance of breastfeeding can reduce transmission of HIV, but the vertical transmission rate was unaffected by their use in our study and it remains high in comparison with rates reported from other studies.

Screening and treatment for cervical intraepithelial neoplasia (CIN) in HIV-infected women.

BACKGROUND: To determine: 1) whether the pathology correlates with the degree of immunosuppression, 2) whether there is a relation between pathology and antiretroviral therapies, 3) whether Papanicolau (Pap) smears correlate with colposcopic and histologic findings, 4) whether there is rapid genital disease progression after standard gynaecologic care. METHODS: Retrospective study. Immunologic, gynaecologic and virologic data were extracted either from patients charts or from laboratory testing. RESULTS: At first visit Pap smears resulted normal in 43.7% of the women, 8.4% of the patients had reactive and reparative changes, 2.8% atypical cells of undetermined significance, 33.8% low-grade squamous intraepithelial lesions and 11.3% high-grade squamous intraepithelial lesions. Patients with a normal PAP smear had higher CD4 cell count (318+/-191 cells/mL) compared to patients with squamous intraepithelial lesions (297+/-116 cells/mL) but the difference was not statistically significant (Mann-Whitney test). The distribution of cervical dysplasia was found to be similar regardless of antiretroviral therapy (chi(2) test). The sensitivity and specificity of Pap tests for detecting CIN were 94 and 80%. Twenty-two per cent of surgically treated women had persistent or recurrent disease. CONCLUSIONS: Lower CD4+ cell counts are not predictive of the presence of cervical dysplasia. All HIV-infected women, independently from their immunological and clinical conditions, need regular Pap smears with appropriate follow-up for abnormal cervicovaginal cytology, this could prevent nearly all deaths from cervical cancer.

Novel 7-oxyiminomethyl derivatives of camptothecin with potent in vitro and in vivo antitumor activity.

In an attempt to synthesize potential anticancer agents acting by inhibition of topoisomerase I (Topo I) a new series of oxyiminomethyl derivatives in position 7 of camptothecin (CPT) was prepared. The synthesis relied on the condensation of 20S-CPT-7-aldehyde or 20S-CPT-7-ketones with alkyl, aryl, heteroaryl, arylalkyl, and heteroarylalkyl O-substituted hydroxylamines. The compounds were tested for their cytotoxic activity in vitro against H460 non-small lung carcinoma cell line, the activity being for 24 out of 37 compounds in the 0.01-0.3 microM range. A QSAR analysis indicated that lipophilicity is the main parameter correlated with cytotoxicity. Investigation of the DNA-Topo I-drug cleavable complex showed a rough parallelism between cytotoxicity and inhibition of Topo I. Persistence of the DNA cleavage after NaCl-mediated disruption of the ternary complex suggests that for the most potent compounds, e.g., 15, the cytotoxicity was at least in part related to stabilization of the complex, as also supported by the persistence of the DNA-enzyme complex in drug-treated cells. The in vivo antitumor efficacy of the most potent analogue (15) was evaluated in direct comparison with topotecan using human lung tumor xenograft models. In the range of optimal doses (2-3 mg/kg), the improved efficacy of 15 was documented in terms of inhibition of tumor growth and rate of complete response.