Gran mejora en artrosis mediante el Servicio Profesional Farmacéutico Asistencial (SPFA) de Ayuda al Tratamiento del Paciente con Dolor (ATRAPADOL) / Great improvement in osteoarthritis through the Professional Pharmaceutical Assistance Service (SPFA) for the Treatment of Pain Patients (ATRAPADOL)

PRESENTACIÓN DEL CASO: mujer de 53 años, diagnosticada de artrosis con dolor de rodillas de más de 6 semanas de duración, diabética, sobrepeso tipo 2 y en tratamiento con diclofenaco y paracetamol, entra en el Servicio ATRAPADOL. Evaluación: se recogen los siguientes datos: Índice Masa Corporal (IMC) =28’1, Perímetro abdominal=97cm, Presión Arterial=135/89 mmHg, HbA1c=6’8, Ejercicio (camina 30min diarios x 2 días) y No fumadora. Escala Visual analógica (EVA)= 8, Índice Lattinen=9, Cuestionario WOMAC=42 y diario del dolor. Además, se registra la medicación prescrita, paracetamol 1g (1-0-0) y diclofenaco 50mg (0-0-1). Se valora la Adherencia Terapéutica con el Test de Hayness-Sackett (Sí cumplidora). INTERVENCIÓN: derivación al médico por HbA1C = 6’8. Servicio ATRAPADOL en 4 visitas: 24 noviembre, 1 y 21 diciembre 2021 y 24 febrero 2022 con las siguientes actuaciones: Indicación Farmacéutica con Colágeno tipo II, Cúrcuma y Ácido Hialurónico + crema efecto frío con condroitina para mejorar el dolor articular, medidas no farmacológicas y derivación al Servicio de Consejo Nutricional.RESULTADOS: la evolución de la paciente de la primera visita, el 24 noviembre 2021 a la tercera visita, el 21diciembre 2022 fue la siguiente: EVA pasó de un valor de 8 a uno de 2, el índice de Lattinen pasó de un valor de 9 a uno de 4. El cuestionario WOMAC pasó de un valor de 42 (dolor=14, capacidad funcional =26 y rigidez=2) a un valor de 5 (dolor=2, Capacidad Funcional=2 y rigidez= 1). En la tercera visita finalizó terapia con diclofenaco y paracetamol. En la cuarta visita del 24 febrero: el IMC pasó de 28,1 a 26,8 (de soprepeso tipo 2 a tipo1). El perímetro abdominal: de 97 a 90 cm; La HbA1c pasó de 6,8 a 6,1. Presión Arterial de 135/89 a 118/83 mmHg. En la cuarta visita refiere ejercicio 30 minutos diarios, seis días a la semana. Los resultados obtenidos son estadísticamente significativos y demuestran mejora en dolor y calidad de vida. (AU)

Total body irradiation using helical tomotherapy: Set-up experience and in-vivo dosimetric evaluation.

PURPOSE: Helical Tomotherapy (HT) appears as a valuable technique for total body irradiation (TBI) to create highly homogeneous and conformal dose distributions with more precise repositioning than conventional TBI techniques. The aim of this work is to describe the technique implementation, including treatment preparation, planning and dosimetric monitoring of TBI delivered in our institution from October 2016 to March 2019. MATERIAL AND METHOD: Prior to patient care, irradiation protocol was set up using physical phantoms. Gafchromic films were used to assess dose distribution homogeneity and evaluate imprecise patient positioning impact. Sixteen patients' irradiations with a prescribed dose of 12Gy were delivered in 6 fractions of 2Gy over 3 days. Pre-treatment quality assurance (QA) was performed for the verification of dose distributions at selected positions. In addition, in-vivo dosimetry was carried out using optically stimulated luminescence dosimeters (OSLD). RESULTS: Planning evaluation, as well as results of pre-treatment verifications, are presented. In-vivo dosimetry showed the strong consistency of OSLD measured doses. OSLD mean relative dose differences between measurement and calculation were respectively +0,96% and -2% for armpit and hands locations, suggesting better reliability for armpit OSLD positioning. Repercussion of both longitudinal and transversal positioning inaccuracies on phantoms is depicted up to 2cm shifts. CONCLUSION: The full methodology to set up TBI protocol, as well as dosimetric evaluation and pre-treatment QA, were presented. Our investigations reveal strong correspondence between planned and delivered doses shedding light on the dose reliability of OSLD for HT based TBI in-vivo dosimetry.

Heterogeneous nuclear ribonucleoprotein A2 is a SET-binding protein and a PP2A inhibitor.

The oncoprotein SET participates in a diversity of cellular functions including cell proliferation. Its role on cell cycle progression is likely mediated by inhibiting cyclin B-cdk1 and the protein phosphatase 2A (PP2A). On identifying new SET cellular partners, we found that SET interacts in vitro and in vivo with the heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2); a protein involved in various aspects of mRNA biogenesis. The SET-binding region of hnRNPA2 is the RNP1 sequence that belongs to the RNA-binding domain (RBD) of this protein. We also found that hnRNPA2 has much higher affinity for single-standed DNA than for SET. On analysing the effect of hnRNPA2 on PP2A inhibition by SET, we observed that hnRNPA2 cooperates with SET on PP2A inhibition. This is because we found that hnRNPA2 is also a PP2A inhibitor. HnRNPA2 interacts with PP2A by the RNP1 sequence; however, to inhibit PP2A activity, the complete RBD is needed. We also observed that overexpression of hnRNPA2 inhibits PP2A activity and stimulates cell proliferation. Interestingly, the overexpression of the complete RBD is sufficient to stimulate proliferation. As hnRNPA2 is overexpressed in a variety of human tumors, our results suggest that hnRNPA2 might participate in oncogenesis by stimulating cell proliferation.

Protein phosphatases 1 and 2A promote Raf-1 activation by regulating 14-3-3 interactions.

Raf-1 activation is a complex process which involves plasma membrane recruitment, phosphorylation, protein-protein and lipid-protein interactions. We now show that PP1 and PP2A serine-threonine phosphatases also have a positive role in Ras dependent Raf-1 activation. General serine-threonine phosphatase inhibitors such sodium fluoride, or ss-glycerophosphate and sodium pyrophosphate, or specific PP1 and PP2A inhibitors including microcystin-LR, protein phosphatase 2A inhibitor I(1) or protein phosphatase inhibitor 2 all abrogate H-Ras and K-Ras dependent Raf-1 activation in vitro. A critical Raf-1 target residue for PP1 and PP2A is S259. Serine phosphatase inhibitors block the dephosphorylation of S259, which accompanies Raf-1 activation, and Ras dependent activation of mutant Raf259A is relatively resistant to serine phosphatase inhibitors. Sucrose gradient analysis demonstrates that serine phosphatase inhibition increases the total amount of 14-3-3 and Raf-1 associated with the plasma membrane and significantly alters the distribution of 14-3-3 and Raf-1 across different plasma membrane microdomains. These observations suggest that dephosphorylation of S259 is a critical early step in Ras dependent Raf-1 activation which facilitates 14-3-3 displacement. Inhibition of PP1 and PP2A therefore causes plasma membrane accumulation of Raf-1/14-3-3 complexes which cannot be activated.

Differential association of p21Cip1 and p27Kip1 with cyclin E-CDK2 during rat liver regeneration.

BACKGROUND/AIMS: The cell cycle inhibitors p21Cip1 and p27Kip1 regulate liver regeneration by modulating the activity of cyclin-dependent kinases (CDKs). However, the specific role of these inhibitors in the regulation of CDK2 activity during liver regeneration remains unknown. The aim of this study was to examine the association of p21Cip1 and p27Kip1 with cyclin E-CDK2 and cyclin A-CDK2 complexes during rat liver regeneration and to correlate the association of both inhibitors with CDK2 activity. METHODS: The association of p21Cip1 or p27Kip1 with cyclin E-CDK2 or cyclin A-CDK2 and the activities of these complexes were analyzed by immunoprecipitation of rat liver homogenates obtained at different times after a partial hepatectomy (PH), followed by Western blotting or kinase assays. RESULTS: High amounts of p27Kip1 bound to cyclin E-CDK2 were observed during the first 13 h after PH, when CDK2 activity was very low. At 24 h, when CDK2 activity was maximal, the amount of bound-p27Kip1 decreased strongly. The amount of p21Cip1 bound to these complexes was low during the first 13 h but subsequently increased. No cyclin A-CDK2 complexes were found during the first 13 h after PH. At 24 h, complexes containing low levels of both inhibitors were detected and at 28 h, a significant increase in p21Cip1 and p27Kip1 associated with cyclin A-CDK2 was observed. CONCLUSIONS: p27Kip1 acts as a brake on cyclin E-CDK2 activity during the first 13 h after a PH. Both p21Cip1 and p27Kip1 down-regulate cyclin A-CDK2 activity at 28 h after PH, after its maximal activation.

The protein SET regulates the inhibitory effect of p21(Cip1) on cyclin E-cyclin-dependent kinase 2 activity.

The cyclin-dependent kinase (CDK) inhibitor p21(Cip1) has a dual role in the regulation of the cell cycle; it is an activator of cyclin D1-CDK4 complexes and an inhibitor of cyclins E/A-CDK2 activity. By affinity chromatography with p21(Cip1)-Sepharose 4B columns, we purified a 39-kDa protein, which was identified by microsequence analysis as the oncoprotein SET. Complexes containing SET and p21(Cip1) were detected in vivo by immunoprecipitation of Namalwa cell extracts using specific anti-p21(Cip1) antibodies. We found that SET bound directly to p21(Cip1) in vitro by the carboxyl-terminal region of p21(Cip1). SET had no direct effect on cyclin E/A-CDK2 activity, although it reversed the inhibition of cyclin E-CDK2, but not of cyclin A-CDK2, induced by p21(Cip1). This result is specific for p21(Cip1), since SET neither bound to p27(Kip1) nor reversed its inhibitory effect on cyclin E-CDK2 or cyclin A-CDK2. Thus, SET appears to be a modulator of p21(Cip1) inhibitory function. These results suggest that SET can regulate G(1)/S transition by modulating the activity of cyclin E-CDK2.

Activation of cdk4 and cdk2 during rat liver regeneration is associated with intranuclear rearrangements of cyclin-cdk complexes.

Partial hepatectomy (PH) triggers the entry of rat liver cells into the cell cycle. The signals leading to cell-cycle activation converge into a family of kinases named cyclin-dependent kinases (cdks). Specific cyclin-cdk complexes are sequentially activated during the cell cycle. Cyclin D-cdk4 and cyclin E-cdk2 are activated during the G1 phase, cyclin A-cdk2 is activated during the S phase, and cyclin B-cdk1 during mitosis. In the present study, we have examined the timing of the activation of cdk4 and cdk2, the intracellular location of G1/S cyclins and cdks, and the relationship between location and cdk4 and cdk2 activities during rat liver regeneration after a PH. Results showed that the activity of both kinases started at 13 hours and showed maximal levels at 24 hours after hepatectomy. In quiescent cells, cyclin D3 and cdk4 were cytoplasmatic, whereas cyclin D1 was nuclear. At 5 hours after hepatectomy, cyclin D3 and cdk4 began to move into the nucleus, and at 13 hours, they were mostly nuclear. During the first 13 hours after hepatectomy, significant amounts of cyclin D1-cdk4 and cyclin D3-cdk4 complexes were formed, but they were mostly inactive. At 24 hours, these complexes were maximally activated. This activation was associated with the accumulation of cyclin D1, cyclin D3, and cdk4 in a nuclear subfraction extractable with nucleases. At 28 hours, the activity of cdk4 in this nuclear subfraction decreased when cyclin D1 moved from this fraction to the nuclear matrix (NM) and the levels of cyclin D3 diminished. The maximal activation of cdk2 at 24 hours was also associated with the accumulation of cyclin E, cyclin A, and cdk2 in this nuclease-sensitive fraction. The inactivation of cdk2 at 28 hours was associated with a strong decrease in cdk2 in this nuclear subfraction. Thus, results reported here indicate that the activation of cdk4 and cdk2 observed in rat liver cells after a PH is associated with a specific intranuclear location of these cdks and their associated cyclins.

Cyclin A is present in the endocytic compartment of rat liver cells and increases during liver regeneration.

Recent studies have implicated the cell cycle kinase cdc2 and cyclin A in the inhibition of the fusion of endocytic vesicles in vitro during mitosis. However, the presence of cyclins or their associated cyclin dependent kinases (cdks) in the endocytic fractions have not been reported. Using Western-blotting and immunocytochemistry approaches with different anticyclin A antibodies we have detected cyclin A in the endocytic compartment of the rat liver. During the pre-replicative phase of liver regeneration the amount of cyclin A in endosomes increases significantly with a peak around 12 hours after partial hepatectomy. Cyclin A-dependent kinases, cdc2 and cdk2, were also found in isolated endosome fractions, showing a distinct kinetics of accumulation during the regenerative period. Finally, histone H1 kinase activity was detected associated with cyclin A in endocytic vesicles and increased in regenerating liver. These results suggest that changes in the organization and in the function of the endocytic compartment during the hepatocellular proliferation may be modulated by proteins involved in the regulation of the cell cycle.

The cell cycle inhibitor p21CIP is phosphorylated by cyclin A-CDK2 complexes.

We report here experimental evidence indicating that the p21CIP, the universal inhibitor of cyclin-dependent protein kinases, general inhibitor CDKs is a substrate oof cyclin A-cdk2. The evidence comes from phosphorylation experiments in which the endogenous p21CIP present in using the original cyclin A-cdk2 complexes immunoprecipitated from HeLa cells extracts can be phosphorylated by the cdk2 of the same complexes. In vitro experiments showing that reconstituted GSTcyclin A-GSTcdk2 complexes from phosphorylate recombinant GSTp21CIP confirms that p21CIP is a cyclin A-cdk2 substrate.

Putative nuclear cdk2 substrates in normal and transformed cells.

The presence of putative substrates of cdk2 in a nuclear fraction obtained by DNase plus RNase extraction (S1 fraction) has been analyzed by immunoprecipitation using specific anti-cdk2 antibodies, followed by phosphorylation assays. S1 nuclear fractions from four different cellular types, two normal (rat hepatocytes and human T lymphocytes) and two transformed (HeLa and Namalwa cells), have been studied. Results indicate that the normal cells share three putative nuclear cdk2 substrates of 21, 37 and 57 kDa. On the other hand, only a substrate of 20 kDa is shared by the two transformed cell lines. On comparing the proliferating normal lymphocytes with the lymphoblastoid cell line Namalwa, it can be observed that they share two proteins of 40 and 70 kDa.

Cyclin/cdk2 complexes in the nucleus of HeLa cells.

Two different fractions of cdk2 and cdc2 have been found in the nucleus of HeLa cells. One, which can be extracted by nuclease treatment, possibly associated with DNA- or RNA-containing structures and another one, which is bound to the nuclear matrix. Nuclear cdk2 forms high molecular weight complexes which migrate at the same position as DNA polymerase alpha and proliferating cell nuclear antigen in sucrose gradient centrifugation experiments. These results suggest that nuclear cdk2 complexes could be associated with the replication factories. Immunoprecipitation experiments reveal that nuclear cdk2 complexes display histone H1-kinase activity and phosphorylate a protein of 18 kDa which is present in these complexes.

Microsomal localization of cyclin A and cdk2 in proliferating rat liver cells.

The expression and intracellular localization of cyclin A and cdk2 have been analyzed in rat liver cells proliferatively activated in vivo by a partial hepatectomy. Western blot analysis revealed that cyclin A started to increase during G1 (at 6 h after hepatectomy) reaching maximal levels during S phase (at 18 h). Cdk2 began to increase during late G1 (at 12 h) peaking also at 18 h. At the latter time cyclin A was mainly localized in the microsomal fraction, although it was also present in cytosol, plasma membrane and nucleus. Active cyclin/cdks complexes containing cyclin A and cdk2 were obtained by precipitation with p13-Sepharose after solubilization of microsomes with triton X-100. The presence of active cyclin A/cdk2 complexes in microsomes was confirmed by immunoprecipitation experiments with anti-cdk2 antibodies. These results suggest a putative role of cyclin A/cdk2 during S phase which would be related with microsomal function.