Osteogenic potential of cells derived from nasal septum.

BACKGROUND: The research addressed to detect new molecular targets in the development of therapeutic strategies aimed to repair bone tissues. The AIM OF THIS STUDY was to determine the potential osteogenic activity of bone cells from the nasal septum and their use to perform accurate molecular analysis from a single sample. METHODOLOGY: The cells, after nasal septum surgery, were subjected to gene silencing, Reverse Transcriptase - Polymerase Chain reactions, immunocytochemistry and chromatin immunoprecipitation. RESULTS: Cells from the nasal septum can give rise to mature osteoblasts that express osteogenic markers (ALP, Runx2, Slug) and are able to mineralize. We demonstrated that Runx2, a transcription factor critical in early osteospecific differentiation, interacts in vivo with the promoter of the SLUG gene, a marker of osteoblast maturation. CONCLUSIONS: We demonstrated that nasal septum-derived osteoblasts represent an interesting alternative source for bone forming cells, and a promising material to be utilized in bone cellular therapy.

Postoperative radiotherapy after radical prostatectomy for prostate carcinoma: the experience of the Brescia Radium Institute.

PURPOSE: The purpose of this study was to evaluate the efficacy of postoperative radiotherapy in reducing the incidence of prostate carcinoma (PCa) recurrences after radical prostatectomy (RP), define the importance of the time interval between surgery and radiotherapy for prognosis and the toxicity of the treatment in comparison with radiotherapy or surgery alone. MATERIALS AND METHODS: We examined 97 patients who consecutively underwent postoperative radiotherapy after RP between 1980 and 2003. The treatment was considered "adjuvant" if was conducted less than 6 months after RP, if there was no macroscopic residual disease and if there was no progressive increase in serum prostate-specific antigen (PSA) and "salvage" if performed more than 6 months after RP, for the presence of macroscopic recurrence or with rising PSA. Radiotherapy was salvage in 56 patients and adjuvant in 41. Age range was 60-70 years in 80% of patients, and the Karnofsky index was over 80 in 78% of cases. Histology revealed extracapsular spread in 60% of patients. Preradiotherapy PSA was higher than 1 ng/ml in 36%. Radiotherapy was performed on the surgical bed only in 80%, and the total dose was 70 Gy in 62% of cases. RESULTS: Recurrence-free survival (RFS) at 5 years and 10 years was 53+/-8% and 32+/-14.2%, respectively, for the whole sample; 76+/-9% and 38+/-2.7% for patients treated with adjuvant radiotherapy and 36+/-10% and 28+/-10% for those treated with salvage radiotherapy (p<0.01). Moreover, the 5-year RFS was better in the group treated with adjuvant radiotherapy and PSA less than or equal to 1 ng/ml (p<0.05). Treatment toxicity was acceptable. CONCLUSIONS: Postoperative radiotherapy improves RFS and reduces the risk of local recurrence. The best results are obtained with early postoperative treatment ("adjuvant"); adjuvant radiotherapy of high-risk forms yields better results if performed with PSA less than or equal to 1 ng/ml.

A possible solvent effect of adenosine diphosphate influences the binding of 1,N6 ethenoadenosine diphosphate to myosin from skeletal muscle.

Skeletal muscle myosin displays two independent and equivalent binding sites for 1,N6 ethenoadenosine diphosphate, with a dissociation constant of 24.7 microM. MgADP, 10 to 40 microM, behaves as a pure competitive type inhibitor (K(SI)=8-9 microM) for the binding of 1,N6 ethenoadenosine diphosphate to skeletal muscle myosin. On the contrary, the inhibition by MgADP, 0.11-1.54 mM, is neither competitive nor non-competitive nor mixed, as is revealed by the analysis with the general kinetic equation (K.J. Laidler, P.S. Bunting, The Chemical Kinetics of Enzyme Action, 2nd ed., Clarendon, Oxford, 1973, p. 94). To explain our finding we propose that MgADP operates a complex type of inhibition, acting both directly as a competitor for myosin active sites, and indirectly by perturbing the regions of the solvent near to the protein.

Dissecting the free energy of formation of the 1:1 actomyosin complex.

The behaviour of solutions of pure myosin, of pure F-actin and of the equimolar mixture of myosin and of F-actin is studied. It is found that the chemical potential of the two proteins, in separate solutions, increases monotonically with the increase of protein osmotic pressure. A method is presented to determine the chemical potential of the 1:1 actin-myosin complex formed from equimolar solutions of myosin and of F-actin (as monomer). This is the first evaluation of the chemical potential of actomyosin under conditions similar to those of skeletal muscle. It is found that the filament suspensions of myosin and of the 1:1 actin-myosin complex display a high non-ideal behavior as well as distinctly different energy profiles as a function of protein osmotic pressure. This supports the hypothesis that, in muscle: (a) detached cross-bridge change significantly their free energy when sarcomere is shifting from the relaxed to the active or to the rigor state; and (b) the cross-bridge attachment-detachment process is accompanied by changes of muscle protein osmotic pressure.

Anomalous binding of MgADP to myosin of skeletal muscle.

Binding of adenosine diphosphate to skeletal muscle myosin was studied using a range of concentrations from 0 to 2 mM. Up to 0.2 mM adenosine diphosphate two equivalent and independent nucleotide binding sites were detected, characterized by the single association constant of 5 x 10(4)M(-1). At greater adenosine diphosphate concentrations a decreasing binding capacity was noticed, bound nucleotide being essentially approximately 0.1 mol/mol at a 1-2mM adenosine diphosphate concentration. We tentatively propose that nucleotides act indirectly on myosin by promoting the perturbation of the solvent, which is supported by the fact that polyphosphates are known powerful kosmotropes.

Quantitative immunoprofiles of breast cancer performed by image analysis.

OBJECTIVE: To determine the biopathologic profiles of breast cancer for greater knowledge of tumor natural history and clinical outcome. STUDY DESIGN: In 99 in situ (ISC) and 2718 infiltrating breast carcinomas (IC), biologic markers (estrogen receptor [ER], progesterone receptor [PR] proliferation index, cerbB-2/NEU, p53, bcl-2 and DNA ploidy) were evaluated with an image analysis system (CAS 200/486). In 105 mixed invasive cancers with size < or = 1 cm, a separate analysis of in situ (ISCm) and invasive component (ICm) was obtained. A clinical study of 836 invasive breast cancers was performed. RESULTS: Different biophenotypes were obtained: among ISCs, cribriform type exhibited biologic behavior similar to that of normal breast tissue (ER+, PR+, proliferation index [PI] low, NEU-, p53-, bcl-2+) the opposite profile was displayed by comedo type, and intermediate phenotypes were observed in noncomedo and lobular types. Comparing ISC and ISCm, PI and p53 expression had the highest levels in ISCm with respect to other groups. NEU overexpression exhibited a decreasing value from ICm to IC. Younger women (< or = 40 years) with IC demonstrated a worse biologic profile (high PI, p53+, ER- and size > 2 cm). In multivariate analysis, PI and NEU in node-negative patients, and NEU, PR and size in node-positive ones emerged as prognostic parameters. CONCLUSION: The results underline the importance of the quantitative biologic profile for defining tumor behavior and patient management.

Protein cross talking through osmotic work: the free energy of formation of the MgADP-myosin complexes at the muscle protein osmotic pressure.

A method is presented to determine the energy of formation of the myosin-ADP complexes at the muscle protein osmotic pressure. It is found that, at 18 kP, the putative protein osmotic pressure in skeletal muscle, the increase of MgADP from 0.05 to 2 mmolal, increases the free energy of myosin-ADP and of myosin-(ADP)2 by 0. 756 and by 9.85 kJ/mol, respectively, and decreases the free energy of myosin by 8.34 kJ erg/mol. It is pointed out that the local changes of water chemical potential, induced by the binding of MgADP to myosin, can be sensed by other structures of the contractile machinery, which per se may even be insensitive to MgADP. Cross talking between macromolecules can thus be achieved by changes of the water chemical potential.

Myofibrils of skeletal muscle: the activity coefficient of orthophosphate.

In the myofibrils of skeletal muscle, at 22 degrees C, pH 7.1 and at the physiological protein osmotic pressure of 1.8 x 10(5) dynes/cm2, orthophosphate behaves quite ideally, the activity coefficient being 0.85. Under the same conditions and at saturation, 2.67 mumoles of orthophosphate are bound per gram of dry myofibrils, with a dissociation constant of 7 x 10(-5) molal. Work is in progress to determine the activity coefficients of adenine nucleotide analogues. This work is needed to assess the actual value of the free energy of hydrolysis of ATP in muscle.

MIB-1 proliferative activity in invasive breast cancer measured by image analysis.

AIMS: To determine cell proliferation in infiltrating breast carcinomas. METHODS: Using the MIB-1 monoclonal antibody, the proliferation index was measured in paraffin wax sections of 871 breast cancers. The MIB-1 proliferation index was compared with other markers of disease progression: size, lymph node status, histotype, oestrogen and progesterone receptor status, expression of p53 and Neu, and DNA ploidy. All parameters were measured using image analysis. In 347 tumours, the MIB-1 and Ki-67 proliferation indexes were compared. Follow up data were available for 170 cases (median 66.5 months). RESULTS: Of the tumours, 314 (36%) had a high proliferation index. The MIB-1 proliferation index was correlated directly with size, nodal status, overexpression of p53 and Neu, and the DNA index; and inversely with oestrogen and progesterone receptor status. The correlation between MIB-1 and Ki-67 proliferation indexes was statistically significant. In patients with pT1 tumours, a low proliferation index correlated with a longer relapse-free interval and overall survival; node negative patients with a low proliferation index had a longer overall survival. CONCLUSIONS: The MIB-1 proliferation index is a reliable, practical and useful method of measuring proliferative activity and is an important predictor of clinical behaviour.

Inhibitory effect of dihydrotestosterone on human thyroid cell growth.

Sex steroid-binding activities have been identified by several authors in normal and pathological thyroids and the expression of the canonic androgen receptor (AR) has recently been demonstrated in human thyroid follicular cells. In order to assess what influence, if any, androgen exposure has on thyroid cell growth, the effect of dihydrotestosterone (DHT) on [3H]thymidine (thy) incorporation and cell proliferation was investigated in thyroid follicular cells in vitro. In a primary culture of goitrous cells, DHT induced a significant reduction of [3H]thy incorporation at concentrations ranging from 10(-12) to 10(-8) M, with a more pronounced effect at 10(-9) M. At this concentration, the inhibitory effect was evident after both 24 and 48 h of treatment and in various types of primary thyroid cell cultures. In goitrous cells, the DHT-induced decrease of [3H]thy was associated with a reduction of expression of the proliferation-associated nuclear Ki-67 antigen, a protein commonly used to assess cell growth fraction. In TPC cells, an AR-positive thyroid papillary carcinoma cell line, DHT at concentrations between 10(-12) and 10(-8) M significantly decreased the growth rate. DHT (10(-9) M) produced an approximately 50-60% inhibition of cell proliferation and the antiandrogen cyproterone acetate was capable of reversing such effects. The DHT-induced reduction of TPC cell proliferation was associated with a significant reduction of c-myc RNA levels. Thyroperoxidase mRNA levels and thyroglobulin production were not reduced by androgen in primary cultures of goitrous cells. In conclusion, our results indicated that androgens may have a role in this gland by reducing the proliferation, but not the function, of follicular cells.

A model relating protein osmotic pressure to the stiffness of the cross-bridge components and the contractile force of skeletal muscle.

We have modeled the effect of protein osmotic pressure on the orientation of the monomer in F-actin, in tropomyosin-F-actin, in the myosin subfragment-1 decorated F-actin and in the myosin subfragment-1 decorated tropomyosin-F-actin. According to the model, at the physiological protein osmotic pressure (18 kPa), the elastic moduli by bending of the monomer in F-actin and in tropomyosin-F-actin are calculated to be 4.74 MPa and 5.8 MPa, respectively. The elastic moduli by bending of the monomer in the myosin subfragment-1 decorated F-actin and in the myosin subfragment-1 decorated tropomyosin-F-actin are calculated to be 22MPa and 22.3MPa, respectively. These latter values are in excellent agreement with the values of the elastic moduli by stretching found for the fibres of frog and rabbit muscle. We have also calculated that, at the physiological protein osmotic pressure, the myosin subfragment-1 decorated F-actin rigor complex can develop a force of 3.96 pN, a force correctly oriented to promote the sliding of the actin filament toward the center of the sarcomere. The magnitude of this force is comparable to that reported for intact skeletal muscle. In contrast, the myosin subfragment-1 decorated tropomyosin-F-actin rigor complex develops a much smaller driving force, that favours relaxation. Apparently tropomyosin uncouples the osmotic and the mechanical event. It is proposed that the elastic energy for muscle contraction is provided by protein osmotic pressure.

The osmotic properties and free energy of formation of the actomyosin rigor complexes from rabbit muscle.

We have studied the osmotic properties of the calcium-regulated actomyosin complexes from skeletal muscle at the protein osmotic pressure of 18 kPa and a different actin-to-myosin molar ratios. Essentially, protein solutions were equilibrated against a solution of poly(ethylene glycol) 40,000 of known macromolecular osmotic pressure. At the end of the equilibration the water and the protein masses of the protein solutions were determined gravimetrically and the protein molar concentration was calculated. In this reconstructed system we have found following, at the actin-to-molar ratio of 2.6 (the most likely stoichiometry of these two proteins in the dense region of the A band) the average distance between the myosin filaments is 34.2 nm, this equals the interfilament distance in the intact fibre of muscle in rigor, at the sarcomere length of 3.38 micrograms. The formation of the F-actin-myosin and of the tropomyosin-F-actin-myosin rigor complexes involves the largest free energy changes, -5.38 kJ/mol myosin and -5.67 kJ/mol myosin, respectively. The formation of the troponin-tropomyosin-F-actin-myosin(Ca) rigor complex from myosin and troponin-tropomyosin-F-actin(Ca) occurs with the free energy change of -3.43 kJ/mol myosin. Of these -3.43 kJ, -1.81 kJ are provided by the endergonic conversion of troponin-tropomyosin-F-actin(EGTA) into troponin-tropomyosin-F-actin (Ca). The transition of myosin and of troponin-tropomyosin-F-actin(EGTA) into the -F-actin-myosin(Ca) rigor complex is accompanied by a 5.8% increase of volume. The increase of volume is due to a large influx of water, which is essentially protein-hydration water.

The stiffness of the crossbridge is a function of the intrinsic protein osmotic pressure generated by the crossbridge itself.

A model is presented that makes it possible to determine the stiffness of the crossbridge from protein osmotic stress experiments. The model was elaborated while studying the osmotic properties of F-actin and of myosin subfragment-1 F-actin. These studies showed that the elastic modulus by bending of the monomer is directly related to the intrinsic protein osmotic pressure of the system. At a protein osmotic pressure of 1.8 x 10(5) dynes/cm2, the physiological protein osmotic pressure of frog skeletal muscle, it was found that the elastic moduli by bending of the monomer in F-actin and in the myosin subfragment-1 decorated F-actin are 6.5 X 10(7) and 3.3 X 10(8) dynes/cm2, respectively. The value of the elastic modulus by bending of the monomer in the myosin subfragment-1 decorated F-actin compares favorably with the values of the elastic modulus by stretching determined in skeletal muscle fibres.

Biophenotypes of breast carcinoma in situ defined by image analysis of biological parameters.

In 50 in situ breast cancers an immunohistochemical study, evaluating estrogen (ER) and progesterone (PR) receptors, Proliferation Index (PI), c-erbB-2/Neu and p53 expression was performed. According to histopathological diagnosis, cases were classified as follows: 14 comedo, 8 solid, 5 micropapillary, 6 lobular, 3 papillary, 1 apocrine and 12 mixed in situ carcinomas. The quantitation of immunohistochemical results was obtained with an image analysis computerized system (CAS 200) with a lesion-field method; tumors were subdivided in fields (1177) histologically homogeneous, with 40 x microscopic objective. For ER, PR, Neu and p53, 10% of the positive area was used as cut-off value; 13% was used for PI. Cribriform and lobular types showed a higher positivity for ER (92.1% and 95.5% of the fields); cribriform and papillary a higher for PR (92.6% and 93.9%). Comedo variant demonstrated the higher PI (52.7%), Neu and p53 expression (67.7% and 43%). A cluster analysis performed on 608 fields, defined two groups according to biological homogeneous criteria. The results obtained identify the different biophenotypes of in situ carcinomas, suggesting the possibility of multiple cancerogenetic ways with a different weight of biological events.

Evidence for androgen receptor gene expression in human thyroid cells and tumours.

Androgen-binding activity has been identified in normal and pathological thyroids, but evidence for the expression of the canonic androgen receptor (AR) in the thyroid has not been provided so far. In this study we have used reverse transcription (RT)-PCR to examine RNA expression of the canonic AR gene in human thyroid tissues, in primary cultures of human thyrocytes and in a variety of neoplastic thyroid cell lines (NPA, TPC and WRO). An AR cDNA fragment with the expected size of 262 bp was detected in normal tissues and cultured thyrocytes as well as in neoplastic cell lines, demonstrating that the gene for AR is indeed expressed in thyroid follicular cells. Immunocytochemical analysis revealed the presence of the AR protein in cancer cell lines and androgen treatment increased nuclear positivity to AR. In a survey of 35 thyroid tissues AR cDNA was detected in all the non-neoplastic samples (6 normal and 3 goitrous) and in 19 of 26 neoplastic samples. AR cDNA was not detected in 4 of the 9 follicular adenomas and in 3 of the 12 papillary carcinomas. AR was revealed by immunohistochemistry in 1 of 2 normal thyroids, in 1 goiter and in 1 of 2 neoplastic thyroids. These findings show the presence of the canonic AR in the human thyroid.

Application of quantitative analysis to biologic profile evaluation in breast cancer.

BACKGROUND: The biologic profile of 907 infiltrating breast carcinomas was determined considering estrogen receptor (ER) and progesterone receptor (PR), proliferation index (PI) and c-erbB-2/Neu expression. The relationship with pathologic parameters (lymph node status, size, histotype) were studied by a multivariate analysis. The clinical prognostic power of biologic profile also was evaluated for 265 patients. METHODS: In 907 infiltrating breast carcinomas, the quantitation of ER, PR, an PI was obtained with an image analysis system (CAS 200, Becton Dickinson Cell Analysis Systems, San Jose, CA); Neu was evaluated semiquantitatively. A clinical study of 265 patients was performed (median follow-up, 42.5 months). RESULTS: Seventy-seven percent of tumors were ER-positive, 70% were PR-positive, 58% had a high PI, and 35% were Neu-positive. The overall analysis indicated a direct correlation between ER and PR (Spearmans' rho [rs] = 0.47, P < 0.001) and an inverse correlation between PI and ER (rs = -0.39, P < 0.001), PI and PR (rs = -0.32, P < 0.001), Neu and ER (rs = -0.20, P < 0.001), and Neu and PR (rs = -0.21, P < 0.001). Cluster analysis, performed based on the biologic profile (ER, PR, PI, c-erbB-2/Neu expression), identified two final groups of tumors with different pathologic features. This study showed a longer relapse free interval for patients with ER- and PR- positive tumors (P = 0.016 and P = 0.007) and low PI and Neu-negative tumors (P < 0.001 and P = 0.047). CONCLUSIONS: These results stress the importance of the biologic profile for defining tumor behavior and patient management, leading to integration of, and eventually the substitution for, the actual staging system.

Osmotic properties of myosin subfragment 1: implications of the mechanism of muscle contraction.

The osmotic behavior of myosin subfragment 1 was studied at 22 degrees C and pH 7.45 in 0.1 m KCl, 2 mm MgCl2, and 10 mm triethanolamine or in 25 mm phosphate, 2 mm MgCl2, and 2 mm MgADP. It was found that, in 0.1 m KCl, myosin subfragment 1 behaved as a spheroidal particle, with an average diameter of 8.09 nm, composed of two myosin subfragment 1 molecules. The lower limit of the thermodynamic dimerization constant was estimated to be 3.5 x 10(4) M-1. Above 5 mm as monomer, myosin subfragment 1 departed from the behavior expected of a dimeric spheroidal model because of the onset of a "hydration force." This force measured at the contact distance between particles equals 2.18 x 10(7) dynes/cm2 and falls off exponentially with a decay distance of 0.27 nm. In 25 mm orthophosphate, myosin subfragment 1, with an increase in the protein osmotic pressure, shifted from the behavior of a sphere to that of a cylinder. Between 1 x 10(5) and 4 x 10(5) dynes/cm2, the behavior of myosin subfragment 1 was different in the presence and in the absence of MgADP. In particular, at 1.8 x 10(5) dynes/cm2, the protein osmotic pressure in frog muscle, myosin subfragment 1 behaved as a sphere of 3.21-nm radius in the presence of MgADP and as a cylinder with a length to diameter ratio of 2.07 in the absence of MgADP. Under the solution conditions used in this work, S1 never behaved as a fully extended particle.

The "in vitro motility assay" and phalloidin-F-actin.

We have compared the osmotic properties of the hydrated, native actin filament and of hydrated phalloidin-F-actin. We have found that phalloidin-F-actin interacts much more strongly with water than native F-actin. It is therefore very likely that the interaction with myosin (that requires the expulsion of the protein solvation water) is more problematic for phalloidin-F-actin that for native F-actin. We conclude that phalloidin-F-actin is not a bona fide substitute for native F-actin in the "in vitro motility assay".