C. Melguizo

Regulatory systems in bone marrow for hematopoietic stem/progenitor cells mobilization and homing.

Regulation of hematopoietic stem cell release, migration, and homing from the bone marrow (BM) and of the mobilization pathway involves a complex interaction among adhesion molecules, cytokines, proteolytic enzymes, stromal cells, and hematopoietic cells. The identification of new mechanisms that regulate the trafficking of hematopoietic stem/progenitor cells (HSPCs) cells has important implications, not only for hematopoietic transplantation but also for cell therapies in regenerative medicine for patients with acute myocardial infarction, spinal cord injury, and stroke, among others. This paper reviews the regulation mechanisms underlying the homing and mobilization of BM hematopoietic stem/progenitor cells, investigating the following issues: (a) the role of different factors, such as stromal cell derived factor-1 (SDF-1), granulocyte colony-stimulating factor (G-CSF), and vascular cell adhesion molecule-1 (VCAM-1), among other ligands; (b) the stem cell count in peripheral blood and BM and influential factors; (c) the therapeutic utilization of this phenomenon in lesions in different tissues, examining the agents involved in HSPCs mobilization, such as the different forms of G-CSF, plerixafor, and natalizumab; and (d) the effects of this mobilization on BM-derived stem/progenitor cells in clinical trials of patients with different diseases.

Inverse expression of mdr 1 and c-myc genes in a rhabdomyosarcoma cell line resistant to actinomycin d.

Cytotoxic agents used in cancer therapy may induce differentiation in tumour cells with no proliferative potential. However, chemotherapy can also induce multidrug resistance, a formidable obstacle to the successful treatment of tumours. Both events were recently shown to occur in a rhabdomyosarcoma cell line (RD‐DAC) resistant to actinomycin D, a drug of choice in the treatment of these tumours. To analyse this connection, cell line RD cultures were investigated with progressive concentrations of actinomycin D and it was shown that a minimum dose (1·2×10−6u2009mm) of the drug was necessary to increase mdr 1 mRNA in RD‐DAC. The mechanism of mdr 1 overexpression was an increase in the number of copies of the mdr 1 gene, although the mRNA levels were not correlated with mdr 1 amplification. Drug resistance mediated by mdr 1 overexpression coincided with the development of myogenic differentiation in RD‐DAC and with a decrease in c‐myc mRNA levels, whereas levels of N‐myc mRNA showed no modulation. These findings suggest that factors implicated in cell proliferation and differentiation, such as c‐myc, may be responsible for the control of genes related to the development of multidrug resistance in rhabdomyosarcomas. Modulation of these factors may determine the sensitivity of rhabdomyosarcoma cells to drugs and may play an important role in triggering the differentiation programme found in these resistant rhabdomyosarcoma cells.

Therapeutic differentiation in a human rhabdomyosarcoma cell line selected for resistance to actinomycin D

Classical cytotoxic treatment of rhabdomyosarcoma (RMS) is accompanied often by significant morbidity and poor response. The use of cytotoxic agents may induce a multidrug resistance phenotype, which plays an important role in the sensitivity of tumoral cells to drugs. Using actinomycin D, a drug of choice in the treatment of RMS, we induced resistance in the TE.32.7 human RMS cell line. The TE.32.7-DAC-resistant cell line exhibited cross-resistance to vincristine and doxorubicin and showed mdr1/P-glycoprotein over-expression, suggesting that this mechanism was involved in the reduction in intracellular drug concentration and may be responsible for the failure of treatment of RMS with classical cycles of cytotoxics. Furthermore, this resistant cell line showed increased expression of the muscle differentiation markers desmin and alpha-actinin and ultrastructural changes which clearly indicated myogenic differentiation. Our findings suggest that, although this tumor is probably arrested along the normal myogenic pathway to maturation, induction of cell differentiation with anti-neoplastic drugs may be an alternative therapeutic approach. However, the failure of TE.32.7-DAC cells to completely re-enter the program of myogenic differentiation supports the hypothesis that multidrug resistance is a major obstacle in differentiation therapy for RMS.

Colon Cancer Therapy: Recent Developments in Nanomedicine to Improve the Efficacy of Conventional Chemotherapeutic Drugs

The number of patients with colorectal cancer, the third most frequently diagnosed malignancy in the world, has increased markedly over the past 20 years and will continue to increase in the future. Despite recent advances in chemotherapy, currently used anticancer molecules are unable to improve the prognosis of advanced or recurrent colorectal cancer, which remains incurable. The transport of classical drugs by nanoparticles has shown great promise in terms of improving drug distribution and bioavailability, increasing tissue half-life and concentrating anticancer molecules in the tumor mass, providing optimal drug delivery to tumor tissue, and minimizing drug toxicity, including those effects associated with pharmaceutical excipients. In addition, colon cancer targeting may be improved by incorporating ligands for tumor-specific surface receptors. Similarly, nanoparticles may interact with key drug-resistance molecules to prevent a reduction in intracellular drug levels drug. Recently published data have provided convincing pre-clinical evidence regarding the potential of active-targeted nanotherapeutics in colon cancer therapy, although, unfortunately, only a few of these therapies have been translated into early-phase clinical trials. As nanotechnology promises to be a new strategy for improving the prognosis of colon cancer patients, it would be very useful to analyze recent progress in this field of research. This review discusses the current status of nanoparticle-mediated cancer-drug delivery, the challenges restricting its application, and the potential implications of its use in colon cancer therapy.

Modulation of α-Actin and α-Actinin Proteins in Cardiomyocytes by Retinoic Acid during Development

Early heart development is known to be sensitive to retinoid concentrations. Although the influence of retinoids on cardiac morphogenesis has been described previously, the effect of retinoids on cardiomyocyte differentiation during development has not been characterized. We quantified the effects of the retinoic acids all-trans RA and 13-cis RA on α-actin and α-actinin at the subcellular level in cultures of chick embryo cardiomyocytes obtained from Hamburger and Hamilton’s (HH) stage 22, 32 and 40 embryos. The retinoids increased the concentration of α-actin and α-actinin in the cytoplasmic and cytoskeletal fractions of cells at all three stages of development. The effect was greatest in cardiomyocytes treated for 24 h with all-trans RA and in cells from HH22 embryos. The greatest increases in α-actin concentration occurred in the cytoskeletal fraction of HH22 cells cultured for 24 h with all-trans or 13-cis RA, whereas the greatest increases in α-actinin were found in the cytoplasmic fraction of HH22 cells exposed to retinoids for 24 h. We conclude that retinoic acid plays a role in the reorganization of the pattern of sarcomeric protein expression during cardiomyocyte differentiation.

Modulation of contractile protein troponin-T in chick myocardial cells by basic fibroblast growth factor and platelet-derived growth factor during development.

We quantified the effect of basic fibroblast growth factor (bFGF) and platelet-derived growth factor (PDGF) on the contractile protein troponin-T (TnT) at the cellular and subcellular level in cultures of chick embryo cardiomyocytes obtained from Hamburger and Hamiltons (HH) stage 19, 29, and 39 embryos. Because expression of thin-filament molecules is considered a good marker of differentiation in muscle cell cultures, we analyzed the modifications these growth factors induced in the transcription of the gene for chick cardiac TnT. Sodium dodecyl sulfate-polyacrylamate gel electrophoresis (SDS-PAGE) and immunoblotting showed that cytoplasmic and cytoskeletal concentrations of TnT are dependent on the stage of embryonic development analyzed and on the type of growth factors added to the culture. The most significant finding was the increase in TnT expression in the cytoplasmic compartment (p < 0.001), accompanied by a slight increase in TnT mRNA, after treatment with bFGF of chick heart cells obtained at HH stage 19. At HH stage 39, bFGF induced less marked changes in the accumulation of TnT in comparison with untreated cardiomyocytes. Our findings support the hypothesis that bFGF plays a role in cardiomyocyte differentiation during early stages of development.

Reverse transcriptase-polymerase chain reaction detection of circulating tumor cells in patients with melanoma: Correlation with clinical stage, tumor thickness and histological type

The aim of this study was to evaluate the correlation between the detection of circulating melanoma cells and prognostic criteria for malignant melanoma such as clinical stage, tumor thickness and histological type of the primary tumor. Using a reverse transcriptase–polymerase chain reaction (RT–PCR) technique, melanoma cells were identified by detecting tyrosinase mRNA in peripheral blood from 58 patients with malignant melanoma classified according to the American Joint Committee on Cancer guidelines. The results of the RT–PCR assay for tyrosinase were related to two prognostic markers typically used to evaluate these tumors: clinical stage and thickness. Positive PCR results were more frequent in primary tumors measuring >u20034u2003mm (83%) than in thinner tumors (1.1–4.0u2003mm, 74%; ≤u20031.0u2003mm, 23%) (Pu2003=u20030.005). No statistical correlation was found between the PCR results and histological appearance of the primary tumor. Although further studies are necessary, our results suggest the possible application of the PCR assay for tyrosinase mRNA in clinical evaluation of the prognosis of malignant melanoma.

Development of Chick Cardiomyocytes: Modulation of Intermediate Filaments by Basic Fibroblast and Platelet-Derived Growth Factors

Recent studies suggest that peptide growth factors play a functional role in cardiac muscle. To test whether embryonic cardiac muscle is a target for regulation by basic fibroblast growth factor and platelet-derived growth factor, we analyzed the effects of these peptides on the expression of the intermediate filaments desmin and vimentin at the subcellular level during development. Sodium dodecyl sulfate-gel electrophoresis, immunoblotting and fluorescence-activated cell sorting analysis were used to study the effect of basic fibroblast growth factor and platelet-derived growth factor on cultures of chick cardiomyocytes during development. Cytoplasmic and cytoskeletal concentrations of desmin and vimentin were dependent on the stage of embryonic development and on the type of growth factor added to the culture. The most significant finding was the increase in desmin expression in the cytoplasmic and cytoskeletal compartments after treatment with basic fibroblast growth factor (10 ng/ml) of chick heart cells at Hamburger and Hamilton stage 19. In more mature stages, basic fibroblast growth factor did not modify the levels of desmin expression. However, this factor led to a progressive deceleration in the rate of increase in vimentin expression. Platelet-derived growth factor increased vimentin expression in all stages studied, the greatest increases appearing in early stages of heart development. Our findings support the hypothesis that basic fibroblast growth factor plays a role in cardiomyocyte differentiation during the early stages of development, whereas platelet-derived growth factor has a dedifferentiating effect.

How is gene transfection able to improve current chemotherapy? The role of combined therapy in cancer treatment.

Despite advances in cancer treatment, a large number of patients eventually develop metastatic disease that is generally incurable. Systemic chemotherapy remains the standard treatment for these patients. Several chemotherapeutic combinations have proven effective in the management of cancer. Paradoxically, although the purpose of polychemotherapy is to improve the prognosis and prolong the survival of patients, it often carries considerable toxicity that causes substantial adverse symptoms. For this reason, a major goal of cancer research is to improve the effectiveness of these cytotoxic agents and reduce their adverse effects. Gene transfer has been proposed as a new strategy to enhance the efficacy of anti-tumor drugs in the treatment of intractable or metastatic cancers. In fact, the association of gene therapy and drugs (combined therapy) has been reported to increase the anti-proliferative effect of classical treatments in lung, bladder, pancreatic, colorectal and breast cancers, among others. Various especially promising therapies have been proposed in this context, including the use of suicide genes, antisense oligonucleotides, ribozymes and RNA interference. In this chapter, we review recent progress in the development of novel anti-cancer strategies that associate cytotoxic agents with gene transfer to enhance their antitumor effect.

Modulation of Myogenic Differentiation in a Human Rhabdomyosarcoma Cell Line by a New Derivative of 5‐Fluorouracil (QF‐3602)

The in vitro study of mechanisms involved in drug‐induced maturation has made it possible to use differentiation‐based therapy in clinical practice. The goal of this new therapy is the development of specific agents to induce cancer cells to stop proliferating and express characteristics of normal cells. Recently, by structural modifications of 5‐fluorouracil (5‐FU), we synthesized a new pyrimidine acyclonucleoside‐like compound, 1‐{[3‐(3‐chloro‐2‐hydroxypropoxy)‐1‐methoxy]propyl}‐5‐fluorouracil (QF‐3602), which showed in rhabdomyosarcoma cells a low toxicity and time‐dependent growth inhibition. In this work, we compared the degree of myogenic differentiation of RD rhabdomyosarcoma (RMS) cells after treatment with QF‐3602 and 5‐FU. Scanning and transmission electron microscopy (SEM and TEM) and immunocytochemical analyses showed that QF‐3602 induced the appearance of myofilaments along the myotube‐like giant RD cells, an increase in fibronectin and a decrease in vimentin expression. In contrast, only minor changes were observed with 5‐FU. Moreover, polymerase chain reaction (PCR) analyses showed that QF‐3602 did not induce overexpression of the mdr 1 gene, a resistance mechanism that frequently appears in classical cytotoxic therapy in these tumors. Compounds obtained by structural modifications of 5‐FU may be useful in differentiation therapy as a new approach to the treatment of RMS.