Synthesis and biological evaluation of dual action cyclo-RGD/SMAC mimetic conjugates targeting α(v)ß(3)/α(v)ß(5) integrins and IAP proteins.

The rational design, synthesis and in vitro biological evaluation of dual action conjugates 11-13, containing a tumour targeting, integrin αvß3/αvß5 ligand portion and a pro-apoptotic SMAC mimetic portion (cyclo-RGD/SMAC mimetic conjugates) are reported. The binding strength of the two separate units is generally maintained by these dual action conjugates. In particular, the connection between the separate units (anchor points on each unit; nature, length and stability of the linker) influences the activity of each portion against its molecular targets (integrins αvß3/αvß5 for cyclo-RGD, IAP proteins for SMAC mimetics). Each conjugate portion tolerates different substitutions while preserving the binding affinity for each target.

Expression of gemcitabine- and cisplatin-related genes in non-small-cell lung cancer.

The aim of this study was to investigate the influence of histology and site of analysis (primary tumor versus lymph node) on the expression of genes involved in gemcitabine and cisplatin activity in non-small-cell lung cancer (NSCLC). Excision repair cross-complementing-1 (ERCC1), human equilibrative nucleoside transporter-1 (hENT1), deoxycytidine kinase (dCK), 5'-nucleotidase (5'-NT), cytidine deaminase (CDA) and ribonucleotide-reductase regulatory subunits (RRM1 and RRM2) were analyzed by quantitative-reverse transcription-PCR in 88 microdissected samples from 69 chemonaive patients. The results showed different patterns of expression for all studied genes, suggesting a possible stratification of the patients. No difference was observed between primary tumor and lymph node metastasis, as well as in adenocarcinoma and squamous-cell carcinoma specimens, while we found a correlation between the CDA-A79C polymorphism and gene expression levels. These data suggest a similar genetic susceptibility to gemcitabine-cisplatin regimens for squamous cell and adenocarcinoma and support the use of both lymph node and primary tumor for the expression profiling of NSCLC.

p27(kip1) acts as a downstream effector of and is coexpressed with the beta1C integrin in prostatic adenocarcinoma.

Integrins are a large family of transmembrane receptors that, in addition to mediating cell adhesion, modulate cell proliferation. The beta1C integrin is an alternatively spliced variant of the beta1 subfamily that contains a unique 48-amino acid sequence in its cytoplasmic domain. We have shown previously that in vitro beta1C inhibits cell proliferation and that in vivo beta1C is expressed in nonproliferative, differentiated epithelium and is selectively downregulated in prostatic adenocarcinoma. Here we show, by immunohistochemistry and immunoblotting analysis, that beta1C is coexpressed in human prostate epithelial cells with the cell-cycle inhibitor p27(kip1), the loss of which correlates with poor prognosis in prostate cancer. In the 37 specimens analyzed, beta1C and p27(kip1) are concurrently expressed in 93% of benign and 84%-91% of tumor prostate cells. Forced expression of beta1C in vitro is accompanied by an increase in p27(kip1) levels, by inhibition of cyclin A-dependent kinase activity, and by increased association of p27(kip1) with cyclin A. beta1C inhibitory effect on cell proliferation is completely prevented by p27(kip1) antisense, but not mismatch oligonucleotides. beta1C expression does not affect either cyclin A or E levels, or cyclin E-associated kinase activity, nor the mitogen-activated protein (MAP) kinase pathway. These findings show a unique mechanism of cell growth inhibition by integrins and point to beta1C as an upstream regulator of p27(kip1) expression and, therefore, a potential target for tumor suppression in prostate cancer.

Monoclonal proliferation of germinal center cells (incipient follicular lymphoma) in an axillary lymph node of a melanoma patient.

A monoclonal proliferation of germinal center cells within a lymph node follicle was incidentally discovered during the staging surgical procedures in a patient with Clark III-level cutaneous melanoma. In one of the 19 axillary lymph nodes examined, we identified a single morphologically atypical lymphoid follicle, predominantly composed of medium-sized cells and immunoreactive for B-cell antigens and for the markers of germinal center origin CD10 and bcl-6. A monoclonal rearrangement of the immunoglobulins heavy chains (IgH) was documented by polymerase chain reaction after laser capture microdissection. The cells of the aberrant follicle expressed the bcl-2 protein at higher levels than the surrounding T lymphocytes in the absence of bcl-2 gene rearrangement. We propose for this lesion the designation of incipient follicular lymphoma. The present findings also confirm the previously reported association between melanoma and lymphoproliferative disorders.

Comparing cephaloauricular and scaphaconchal angles in prominent ear patients and control subjects.

Approximately 5% of 1-year-old children have prominent ears. The most common findings are underdevelopment or lack of the antihelical fold, overdevelopment of the concha, a scapha-conchal angle greater than 130 degrees, and a protruding lobule. This study compared the cephaloauricular and scaphaconchal angles of 15 patients with prominent ears and 15 patients in a control group. Alginate was used to create a mold of each patient's right ear. Afterward the molds were cut transversally for measurement of the angles. The first cut was made at the middle of the ear's cephalocaudal length. The second cut was made in the superior piece midway between the first cut and the superior extremity of the ear. The cephaloauricular angle was defined as the intersection of a straight line running through the tragus insertion and the lateral portion of the mastoid region with a straight line that running through the tragus and the middle of the helix. The scaphaconchal angle was obtained in the second cut by measurement of the angles formed by these two structures molded in the posterior aspect of the ear. The Student's t test was used for statistical analysis. The average cephaloauricular angle was 47.7 degrees for the study group and 31.1 degrees for the control group. The average scaphaconchal angle was 132.6 degrees for the study group and 106.7 degrees for the control group. This study presents a new method for evaluating the angles of the ear, confirming that both measured angles (cephaloauricular and scaphaconchal) are greater in patients with prominent ears (p < 0.005).

Tumour growth fraction and apoptosis in salivary gland acinic cell carcinomas. Prognostic implications of Ki-67 and bcl-2 expression and of in situ end labelling (TUNEL).

bcl-2 protein and Ki-67 (MIB-1) were studied in 32 acinic cell carcinomas (ACCs), all with a minimum of 5 years' clinical follow-up. Tumour apoptosis was evaluated by TdT dUTP nick end labelling (TUNEL) and by morphological criteria. Five patients died of their disease. Patients with stage I tumours had significantly better survival compared with other stages (P < 0.05). Patients with MIB-1-negative tumours had significantly better survival than patients with MIB-1-positive tumours (P = 0.05). This study confirms a previous report that MIB-1 is an independent prognostic factor for survival in patients with ACC. Stage I tumours had high expression of bcl-2 protein, but there was no difference when compared with other stages. TUNEL positivity was most prevalent in stage I tumours, compared with stages II, III, and IV (P < 0.05), probably indicating more apoptosis. This could imply a capacity of stage I tumours ('early tumours') for early selection of tumour cells for elimination by apoptosis. There was no significant difference between expression of bcl-2 and TUNEL, between these parameters and clinical outcome, or between any parameter and morphological subclassification. We conclude that MIB-1 has prognostic value in ACC. Clinical staging, bcl-2, and TUNEL are also potentially useful as prognostic markers.

Down-regulation of beta(1C) integrin in breast carcinomas correlates with high proliferative fraction, high histological grade, and larger size.

beta(1C) integrin is an unspliced form of the integrin beta(1) subfamily, which has been shown to inhibit cell proliferation in vitro. Using an affinity-purified rabbit antibody, we have investigated 283 previously untreated breast carcinomas, with the aim of ascertaining the actual prevalence of beta(1C) expression in these tumors and of defining its pathological correlates. Immunoblotting and reverse transcriptase-polymerase chain reaction experiments have also been performed in selected cases, to confirm the immunocytochemical findings. Overall, beta(1C) immunoreactivity was down-regulated (ie, expressed in < 50% of the neoplastic cells) in 114 cases (40.3%). Down-regulation of beta(1C) expression in breast carcinomas correlated significantly with the tumor grade, the proliferative fraction (as evaluated by Ki-67 immunostaining with the MIB-1 monoclonal antibody), the estrogen and progesterone receptor status, and the tumor size (pT classification) and marginally with the node status. In a multivariate analysis with all available measures fitted simultaneously, tumor grade (P = 0.004), Ki-67 immunolabeling (P = 0.01), and pT categories (P = 0.04) were significantly associated with beta(1C) immunoreactivity. Although the short follow-up time (2-3 years) of the current series of patients does not allow the performance of survival analyses, the correlation of beta(1C) expression with tumor size, grade, and proliferative fraction and its alleged role as an upstream regulator of p27(kip1) make this integrin variant a likely novel prognostic parameter for invasive carcinomas of the breast.

Reverse transcription-polymerase chain reaction assay for multiple mRNA markers in the detection of breast cancer metastases in sentinel lymph nodes.

The identification of specific tumor mRNA markers by reverse transcription-polymerase chain reaction might be a valuable diagnostic adjunct for the detection of breast cancer metastases in axillary sentinel lymph nodes (SLNs). In this study we have compared the diagnostic accuracy of an extensive histopathologic examination of 146 SLNs from 123 breast carcinoma patients with that of the evaluation of 5 mRNA markers. When analyzed individually, none of the different markers attained a sensitivity higher than 77.8%, and the general concordance with the histopathologic findings ranged from 78.8 to 83.6%. In a multiple-marker assay, taking into account the expression of at least 1 of the 5 tumor markers, the sensitivity of the test rose to 95.6%, with a specificity of 66.3% and a general concordance with the histopathologic status of 75.3%. Finally, when at least 2 of 3 markers (maspin, cytokeratin 19 and mammaglobin 1) were expressed, the concordance with either SLN or axillary lymph node status was highest (88.4% and 84.6%, respectively). The high prevalence of positive reverse transcription-polymerase chain reaction assays in histologically uninvolved SLNs, however, may hamper extensive application of these techniques in the clinical setting.

Predictors of survival in subjects with chronic obstructive pulmonary disease treated with long-term oxygen therapy.

We examined 166 patients with advanced chronic obstructive pulmonary disease (COPD) treated with long-term oxygen therapy (LTOT) in order to evaluate the prognostic factors of such patients. The mean observation period was 24 months (range 2-50 months) and the following variables were considered: age, forced expiratory volume in 1 s (FEV1), arterial oxygen tension (PaO2), arterial carbon dioxide tension (PaCO2), hematocrit, right ventricular systolic pressure (RVSP; evaluated by Doppler echocardiography), number of hospitalizations in the 2 years prior to prescription of LTOT and body mass index. The overall survival rate was 78.3% at 24 months and 67.1% at 36 months. A univariate analysis identified three variables as significant predictors of survival: FEV1, PaO2 and RVSP. A multivariate analysis, using Cox's model, showed an independent predictive power for RVSP, age and FEV1. RVSP higher than 35 mm Hg, age greater than 70 years and FEV1 lower than 30% of the predicted value were associated with shortened survival. The importance of pulmonary hypertension as a predictor of death suggests that LTOT could be prescribed earlier for COPD patients with cor pulmonale, as oxygen has been shown to be the only effective therapy for improving the survival probability of these patients.

Beta1C integrin in epithelial cells correlates with a nonproliferative phenotype: forced expression of beta1C inhibits prostate epithelial cell proliferation.

The expression of the beta1C integrin, an alternatively spliced variant of the beta1 subunit, was investigated in human adult and fetal tissues. In the adult, beta1C immunoreactivity was found in nonproliferative, differentiated simple, and/or pseudostratified epithelia in prostate glands and liver bile ducts. In contrast, beta1C was undetectable in stratified squamous epithelium of the epidermis and/or in hepatocytes. Luminal prostate epithelial cells expressed beta1C in vivo and in vitro, but no beta1C was seen in basal cells, which are proliferating cells. Fetal prostate expressed beta1C in differentiated glands that had a defined lumen, but not in budding glands, indicating that beta1C is a marker of prostate epithelium differentiation. The beta1C and the common beta1A variants are differentially distributed: beta1A was found in luminal and basal epithelial as well as in stromal cells in the prostate. In the liver, beta1C and beta1A were coexpressed in biliary epithelium, whereas vascular cells expressed only beta1A. Because we found beta1C in nonproliferative and differentiated epithelium, we investigated whether beta1C could have a causal role in inhibiting epithelial cell proliferation. The results showed that exogenous expression of a beta1C, but not of a beta1A, cytoplasmic domain chimeric construct, completely inhibited thymidine incorporation in response to serum by prostate cancer epithelial cells. Consistent with these in vitro results, beta1C appeared to be downregulated in prostate glands that exhibit regenerative features in benign hyperplastic epithelium. These data show that the presence of beta1C integrins in epithelial cells correlates with a nonproliferative, differentiated phenotype and is growth inhibitory to prostate epithelial cells in vitro. These findings indicate a novel pathophysiological role for this integrin variant in epithelial cell proliferation.