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Undoubtedly, the focus of the field of stem cell research is predominantly aimed at the artificial reprogramming of human somatic cells for the production of induced pluripotent stem (iPS) cells. This relatively new technology may circumvent the ethical issues of using human embryonic stem (hES) cells for the potential applications in cell replacement therapy. Besides such ethical issues, iPS cell technology offers the advantage of obtaining patient-derived tissues and/or cells, which may be utilized for autologous transplantation and tissue regeneration, investigation of a variety of human illnesses and for the screening of new drugs. The field of stem cell research has placed a major emphasis in understanding the genetic and epigenetic codes for pluripotency, in order to control and optimize autologous transplantation techniques and avoid teratoma formation.

The CDK9-related pathway is an important regulator of mammalian cell biology and is also involved in the replication cycle of several viruses, including the human immunodeficiency virus type 1. CDK9 is present in two isoforms termed CDK9-42 and CDK9-55 that bind noncovalently type T cyclins and cyclin K. This association forms a heterodimer, where CDK9 carries the enzymatic site and the cyclin partner functions as a regulatory subunit. This heterodimer is the main component of the positive transcription elongation factor b, which stabilizes RNA elongation via phosphorylation of the RNA pol II carboxyl terminal domain. Abnormal activities in the CDK9-related pathway were observed in human malignancies and cardiac hypertrophies. Thus, the elucidation of the CDK9 pathway deregulations may provide useful insights into the pathogenesis and progression of human malignancies, cardiac hypertrophy, AIDS and other viral-related maladies. These studies may lead to the improvement of kinase inhibitors for the treatment of the previously mentioned pathological conditions. This review describes the CDK9-related pathway deregulations in malignancies and the development of kinase inhibitors in cancer therapy, which can be classified into three categories: antagonists that block the ATP binding site of the catalytic domain, allosteric inhibitors, and small molecules that disrupt protein-protein interactions.

According to National Comprehensive Cancer Network guidelines concerning non–small cell lung cancer, robotic lobectomy is considered an advisable oncologic procedure for patients with lung cancer, with no anatomic or surgical contraindications. At present, da Vinci is the only available robotic system (Intuitive Surgical, Inc., Sunnyvale, CA), with 2 platforms (da Vinci SI and da Vinci XI) requiring different port-mapping and cart positioning. Robotic left lower lobectomy can be considered one of the most technically simple of all lobectomies. We describe our endoscopic technique, with 4 surgical ports, using CO2 insufflation.

Integrating gene delivery systems allow for a more stable transgene expression in mammalian cells than the episomal ones. However, the integration of the shuttle vector within the cellular chromosomal DNA is associated with the risk of insertional mutagenesis, which, in turn, may cause malignant cell transformation. The use of a retroviral-derived vector system was responsible for the development of leukemia in five children, who participated in various clinical trials for the treatment of severe combined immunodeficiency (SCID-X1) in France and in the United Kingdom. Unfortunately, the hematological malignancy claimed the life of one patient in 2004, who was enrolled in the French clinical trial. In addition, adeno-associated-viral-(AAV-) mediated gene transfer induced tumors in animal models, whereas the Sleeping Beauty (SB) DNA transposon system was associated with insertional mutagenesis events in cell culture systems. On these grounds, it is necessary to develop safer gene delivery systems for the genetic manipulation of mammalian cells. This paper discusses the latest achievements that have been reported in the field of vector design.

Background: The incidental detection of solitary pulmonary nodule (SPN) is currently increasing due to the widespread use of computed tomography (CT) during the follow up in oncological patients or in screening trials. A quick and definitive histological diagnosis of these nodes is mandatory as, in case of primitive lung cancer, an early detection could improve both surgical results and prognosis. The minimally invasive pulmonary resection (MIPR) is the gold standard procedure for diagnosis and treatment of small lung nodules, but it can be difficult to localize deep nonpalpable nodes that lie in the lung parenchyma. Hence, throughout the years several techniques have been developed to better localize deep or sub solid nodes. We describe our experience with radio-guided technique. Methods: Patients with SPN smaller than 10 mm and/or with a distance from the visceral pleura equal or larger than 10 mm underwent MIPR after CT injection of a solution containing 0.2 mL 99 Tc-labelled human serum albumin microspheres and 0.1 mL of non-ionic contrast. During surgical procedures, a collimated probe, connected to a gamma-ray detector, was used to scan the lung and detect the target area. The area of major radioactivity was then resected. Results: Between 2010 and 2015, 175 patients (M/F:97/78) underwent minimally invasive resection (thoracoscopic or robotic) with radio-guided technique. The mean node diameter was 13 mm (range, 5–20 mm), and the mean distance from the visceral pleura was 15 mm (range, 6–39 mm). No significant CT-guided-injection-related complications occurred, except for 13 patients (7.4%) who developed a pneumothorax (PNX) not requiring chest tube insertion. This technique guaranteed a successfully intra-operative node localization in 100% of cases. The mean duration of the surgical procedure was 44 min (range, 25–130 min). The mean length of pleural drainage and mean hospital stay was 2.6 days (range, 1–5 days) and 3.9 days (range, 1–7 days) respectively. No mortality or perioperative complications occurred. Pathology reports showed 105 metastases, 55 primitive lung cancers and 15 benign lesions. In case of suspicion of primitive lung tumour an intraoperative frozen exam was conducted, and all patients underwent lobectomy or segmentectomy with lymphadenectomy [41 patients with thoracotomy, 11 with robotic surgery and 3 with video-assisted thoracoscopic surgery (VATS)]. Conclusions: Our experience confirms that radio-guided thoracoscopic surgery (RGTS) is a feasible and safe procedure, thanks to its high success rate the radio-guided technique is helpful to overcome the lack of tactile feedback during MIPR [VATS and robotic-assisted thoracoscopic surgery (RATS)] and to limit conversion to open surgery.

Management of displaced femoral neck fracture in elderly patients is challenging due to the patient's high risk profile, poor quality bone stock and muscle weakness.Fifty-one patients with displaced (Garden 3-4) intracapsular femoral neck fractures were treated with a newly designed Fixion expandable stem hemiarthroplasty implant and followed thereafter for a minimum of 6 months.Thirty patients (58.8%) had concomitant diseases graded 3-4 by the ASA scoring system. Two patients (4.8%) incurred deep wound infections that necessitated hardware removal. One case (2.6%) of dislocation was treated by closed reduction. Forty-two patients (82.4%) survived6 months and cooperated with the study protocol. Mild groin/thigh pain was reported by 11 patients (26.2%). Twenty-six (61.9%) had lost 1-2 out of 4 grades of mobility.Fixion implant may be considered a further step in the evolution of femoral joint hemiarthroplasty. In the short term, it has proven to be as effective as cemented implant and to be user-friendly for the surgeon. It is also anticipated to be feasible whenever revision is required.

Ochratoxin A (OTA) is a natural mycotoxin, involved in the development of important human and animal diseases. In this work we have studied the role of oxidative stress in the development of OTA nephrotoxicity and the effect of a new recombinant mitochondrial manganese containing superoxide dismutase (rMnSOD) to prevent kidney damage induced by OTA. Blood pressure, glomerular filtration rate and renal histology were analyzed in control rats and in OTA treated rats. In addition, lipid peroxidation, catalase and superoxide dismutase productions were measured. Our data showed that animals treated with OTA presented hypertension and reduction of glomerular filtration rate (GFR). These effects are most probably related to an increase in the reactive oxygen species (ROS) productions. In fact, we have shown that treatment with rMnSOD restored the levels of blood pressure and GFR simultaneously. Moreover, we have noted that OTA induced alteration on glomerular and tubular degeneration and interstitial infiltrates and that use of rMnSOD combined with OTA prevent this renal histological damage confirming the potential therapeutic role in the treatment of rMnSOD OTA nephrotoxicity. Ochratoxin A (OTA) is a natural mycotoxin, involved in the development of important human and animal diseases. In this work we have studied the role of oxidative stress in the development of OTA nephrotoxicity and the effect of a new recombinant mitochondrial manganese containing superoxide dismutase (rMnSOD) to prevent kidney damage induced by OTA. Blood pressure, glomerular filtration rate and renal histology were analyzed in control rats and in OTA treated rats. In addition, lipid peroxidation, catalase and superoxide dismutase productions were measured. Our data showed that animals treated with OTA presented hypertension and reduction of glomerular filtration rate (GFR). These effects are most probably related to an increase in the reactive oxygen species (ROS) productions. In fact, we have shown that treatment with rMnSOD restored the levels of blood pressure and GFR simultaneously. Moreover, we have noted that OTA induced alteration on glomerular and tubular degeneration and interstitial infiltrates and that use of rMnSOD combined with OTA prevent this renal histological damage confirming the potential therapeutic role in the treatment of rMnSOD OTA nephrotoxicity.

The Id family of helix-loop-helix proteins is known to be involved in the proliferation and differentiation of several types of cells. The type 1 IGF receptor (IGF-IR) induces either proliferation or differentiation in 32D cells, a murine hemopoietic cell line, depending on the availability of the appropriate substrates for the receptor. We have previously reported that the IGF-IR regulates the expression of the Id2 gene in 32D cells. We now show that the IGF-IR controls the increase in Id2 gene expression through at least three pathways. These three pathways originate from the tyrosine residue at 950, a domain in the C-terminus, and the activation of the insulin receptor substrate-1 (IRS-1) by the receptor. IRS-1 is the preponderant signal, and its effect on Id2 gene expression requires a functional phosphotyrosine binding domain. With wild-type IRS-1, Id2 gene expression is increased, even in those cells that express IGF-I receptors defective in Id2 signaling. Rapamycin, an inhibitor of p70(S6K), a downstream effector of IRS-1 signaling, partially inhibits (but does not completely abrogate) the increase in Id2 gene expression. A mutant IRS-1 with a deletion of the Pleckstrin domain is as effective as wild-type IRS-1 in up-regulating Id2 gene expression. In addition, it seems to increase the stability of p70(S6K). Our results indicate that the IGF-IR regulates Id2 gene expression through different pathways. At least in 32D cells, increased Id2 gene expression seems to correlate more with inhibition of differentiation than with proliferation.

H19-7/IGF-IR cells are rat hippocampal cells expressing a human IGF-I receptor, which differentiate to a neuronal phenotype when stimulated by IGF-I at 39 degrees C. H19-7/IGF-IR cells have low levels of expression of insulin receptor substrate-l (IRS-1), a major substrate of the IGF-IR. IGF-I induces serine-phosphorylation and down-regulation of the endogenous IRS-1 upon differentiation of H19-7/IGF-IR cells. The profound influence of IRS-1 on differentiation of H19-7/IGF-IR cells was confirmed by transfecting these cells with a plasmid expressing mouse IRS-1. Over-expression of wild type IRS-1 in H19-7/IGF-IR cells abolishes IGF-I-induced differentiation at 39 degrees C. A mutant of IRS-1 lacking the PTB domain loses the ability to inhibit the differentiation program. H19-7/IGF-IR/IRS-1 cells at 39 degrees C show a stronger and prolonged activation of Akt, when compared to H19-7/IGF-IR cells. The role of Akt in the inhibition of the differentiation program was confirmed by using the inhibitor of Class I PI3 kinases LY29400, which restores IGF-I-induced differentiation of H19-7/IGF-IR/IRS-1 cells. H19-7/IGF-IR/IRS-1 cells show a strong reduction in MAP kinases signaling, which is related to the superactivation of Akt. This was confirmed by expressing in H19-7/IGF-IR cells a constitutively active Akt, which inhibited MAP kinases activation in these cells. These experiments confirm the importance of MAPK in the mechanism of IGF-I-mediated differentiation of H19-7/IGF-IR cells

LNCaP cells are human prostatic cancer cells that have a frame-shift mutation of the tumor suppressor gene PTEN and do not express the insulin receptor substrate1 (IRS-1), a major substrate of the type 1 insulin-like growth factor receptor (IGF-IR). Ectopic expression of IRS-1 in LNCaP cells increases cell adhesion and decreases cell motility by an IGF-I-independent mechanism. We show now that these eAects of IRS-1 are accompanied by serine phosphorylation of IRS-1 and are inhibited by inhibitors of phosphatidylinositol 3-kinase (PI3K). We have confirmed the requirement for PI3K activity and serine phosphorylation by the use of IRS-1 mutants, expressed in LNCaP cells. Serine phosphorylation inhibits IGF-I-induced tyrosyl phosphorylation of IRS-1, which is restored by the expression of wild-type PTEN or by inhibition of PI3K activity. Finally, IRS-1 in LNCaP cells co-immunoprecipitates with integrin a 5 b 1, and the association is again IGF-I-independent. We conclude that in LNCaP cells, IRS-1 is serine phosphorylated by PI3K, generating eAects that are diAerent, and even opposite, from those generated by IGF-I. Oncogene (2001) 20, 490‐500.

We present a new inflatable self-locking intramedullary nailing system for the treatment of long bone fractures. The features of this system and the advantages of the technique include: fixation along the entire length of the nail, lack of proximal or distal interlocking screws, optional reaming, reduced exposure of the surgeon to X-ray and reduced operating room time. First preliminary clinical and radiographic results are reported. Out of 41 patients who received implants for closed fractures, 29 were available for follow-up. We observed fracture consolidation in all 29 cases (100%), with absence of nonunions, infections or rotational malalignments. Only in 2 (6.9%) cases were complication reported, consisting in the opening of the fracture after inflation of the nail. Good stability of the nail, however, assured a complete callus formation. We conclude that the use of this intramedullary nail is an innovative, effective, simple and minimally invasive treatment for long bone shaft fractures.

LNCaP prostatic cancer cells are characterized by having a PTEN mutation, low levels of type 1 insulin-like growth factor receptor (IGF-IR) and no IRS-1, one of the major substrates of the IGF-IR. The absence of IRS-1, an activator of PI3-kinase, is compensated in these cells by the mutation in PTEN, an inhibitor of PI3-kinase. However, IGF-IR signaling in the absence of IRS-1 can cause cell differentiation and growth arrest. We hypothesized that these three characteristics may not be unrelated, specifically that, together, they may favor the metastatic spread of prostatic cancer cells without decreasing their growth potential. In support of this hypothesis, we report here that: (1) IRS-1 expression increases cell adhesion and decreases cell motility; (2) over-expression of the IGF-IR, in the absence of IRS-1, causes growth arrest and (3) a combination of IGF-IR and IRS-1 restores the transformed phenotype of LNCaP cells. These findings suggest a mechanism by which prostatic cancer cells can achieve metastatic potential without interfering with their growth potential. Oncogene (2000).

Over the last decade, more than 300 phase I and phase II gene-based clinical trials have been conducted worldwide for the treatment of cancer and monogenic disorders. Lately, these trials have been extended to the treatment of AIDS and, to a lesser extent, cardiovascular diseases. There are 27 currently active gene therapy protocols for the treatment of HIV-1 infection in the USA. Preclinical studies are currently in progress to evaluate the possibility of increasing the number of gene therapy clinical trials for cardiopathies, and of beginning new gene therapy programs for neurologic illnesses, autoimmuno diseases, allergies, regeneration of tissues, and to implement procedures of allogeneic tissues or cell transplantation. In addition, gene transfer technology has allowed for the development of innovative vaccine design, known as genetic immunization. This technique has already been applied in the AIDS vaccine programs in the USA. These programs aim to confer protective immunity against HIV-1 transmission to individuals who are at risk of infection. Research programs have also been considered to develop therapeutic vaccines for patients with AIDS and generate either preventive or therapeutic vaccines against malaria, tuberculosis, hepatitis A, B and C viruses, influenza virus, La Crosse virus, and Ebola virus. The potential therapeutic applications of gene transfer technology are enormous. However, the effectiveness of gene therapy programs is still questioned. Furthermore, there is growing concern over the matter of safety of gene delivery and controversy has arisen over the proposal to begin in utero gene therapy clinical trials for the treatment of inherited genetic disorders. From this standpoint, despite the latest significant achievements reported in vector design, it is not possible to predict to what extent gene therapeutic interventions will be effective in patients, and in what time frame.

The clinical efficacy of therapeutic complement (C)-activating monoclonal antibodies (mAb) to melanoma-associated antigens can be impaired by the levels of expression of C-inhibitory molecules on neoplastic cells. Protectin (CD59) is a glycosylphosphatidylinositol (GPI)-anchored cell membrane glycoprotein, acting as terminal regulator of C cascade, which is heterogeneously expressed in melanomas and represents the main restriction factor of C-mediated lysis of melanoma cells. Thus, we investigated whether the overexpression of CD59 could influence the constitutive susceptibility of distinct melanoma cells to homologous C. Infection of CD59-positive Mel 100 and 70-W melanoma cells by a retroviral vector carrying the CD59 cDNA, significantly (P < 0.05) upregulated their constitutive expression of CD59, whereas it did not affect that of additional C-regulatory molecules. Transduced CD59 was entirely GPI-anchored and showed a molecular weight identical to native CD59. Additionally, higher amounts of soluble CD59 were detected in the conditioned media of CD59-transduced melanoma cells compared with parental cells. CD59-transduced melanoma cells, sensitized by the anti-GD3 disialoganglioside mAb R24, were significantly (P < 0.05) less susceptible to homologous C-lysis than were parental cells; this effect was fully reverted by the masking of CD59 with F(ab′)2 fragments of the anti-CD59 mAb YTH53.1. These results provide conclusive evidence demonstrating that absolute levels of CD59 expression regulate the susceptibility to homologous C of specific melanoma cells, and suggest an additional explanation for the poor clinical results obtained with C-activating mAb in the clinical setting. J. Cell. Physiol. 185:317–323, 2000. © 2000 Wiley-Liss, Inc.

HIV-1 gene expression relies upon a complex machinery that is primarily controlled by two viral regulatory proteins, Tat and Rev. Rev is involved in regulating post-transcriptional events of HIV-1 gene expression. The Tat protein transactivates transcription from the HIV-1 5' long terminal repeat (LTR) and acts in synergy with specific cellular factors. Recently, it has been shown that one set of these cellular factors is a protein kinase activity termed TAK (Tat-associated kinase), which activates transcription by hyperphosphorylation of the carboxyl-terminal domain (CTD) of the large subunit of RNA polymerase II. TAK also enhances transcription of HIV-2, together with the retroviral transactivator, Tat-2. The TAK activity appears to be related to the CTD kinase P-TEFb, which stabilizes transcription elongation of many genes and was originally isolated from Drosophila extracts. Both TAK and P-TEFb contain at least two subunits: the cyclin-dependent kinase, CDK9 (PITALRE), the catalytic subunit, and the regulatory subunit, cyclin T1. CDK9 and cyclin T1 are ubiquitous factors that affects many cellular processes, including cell differentiation and apoptosis. The involvement of TAK in HIV-1 and HIV-2 gene expression is an important aspect in the biology of these two retroviruses, and may lead to the development of novel antiretroviral drugs and/or gene therapy approaches for the treatment of patients with AIDS.

Programmed Cell Death (PCD) is known to play an important role in both the development and the growth rate of human tumors. It has in fact been suggested that suppression of the apoptotic pathway is a requirement for the establishment of the transformed phenotype. In order to elucidate the relationship between resistance to apoptosis and transformation, we have asked in this investigation whether or not the two processes can be directly correlated. For this purpose, we have used mouse embryo fibroblasts (MEF) expressing either the wild-type or several mutants of the type 1 insulin-like growth factor receptor (IGF-IR). The wild-type IGF-IR has both transforming and anti-apoptotic activities, and we have asked whether these two activities can be or not separated in mutant receptors. Using this well-defined system, our results show that certain mutants of the IGF-IR that have strong anti-apoptotic and mitogenic activities, are incapable of transforming MEF (colony formation in soft agar). We have, instead, a good correlation between mitogenic and anti-apoptotic activities, suggesting the possibility that the two processes may share similar signaling pathways from the IGF-IR. On the other hand, our results indicate that transformation requires an additional signal, above and beyond the mitogenic and survival signals. Our conclusion is that, at least in this system, the establishment of the malignant phenotype and resistance to apoptosis can be dissociated, implying the possibility of separate targeting. J. Cell. Biochem. 72:294–310, 1999. © 1999 Wiley-Liss, Inc.

The aim of the present study was to externally validate the European System for Cardiac Operative Risk Evaluation (EuroSCORE) II (ESII) in patients undergoing percutaneous coronary intervention (PCI) and to compare its performance with that of its previously released versions, named additive (addES) and logistic EuroSCORE (logES). A total of 537 patients undergoing PCI were analyzed by different measurements of discrimination, calibration, and global accuracy. A significant gradient in all-cause mortality was seen with all the models at 30 days, 1 year, and 5 years, with the exception of the ESII at 30 days. The ESII had the lowest area under the receiver operating characteristic curve at all time points compared with its previous version, being 0.83 (vs 0.90 for both addES and logES) at 30 days, 0.75 (vs 0.82 for both addES and logES) at 1 year, and 0.69 (vs 0.77 for addES and 0.76 for logES) at 5 years. However, the ESII displayed a better calibration than the logES at 30 days, whereas both scores were miscalibrated at 1 and 5 years. The Brier score displayed similar global accuracy between the ESII and logES. In conclusion, the ESII is better calibrated than the logES at 30 days but does not represent a step forward in discrimination and global accuracy compared with its previous versions for predicting early- and long-term mortality of patients undergoing PCI.

Background The aim of the present study was to appraise the comparative ability of different ACEF models incorporating glomerular filtration rate or creatinine clearance estimated by the Modification of Diet in Renal Disease [ACEF MDRD ] or Cokcroft-Gault [ACEF CG ] equations, respectively, over the original ACEF score (ACEF SrCr ) in patients undergoing percutaneous coronary intervention (PCI). Methods A total of 537 patients were analyzed by different measures of discrimination, calibration and net reclassification improvement (NRI). Results A significant gradient in all-cause mortality was consistently seen with all the models at 30days, 1year and 5years. The comparison of the three models showed that the best balance in terms of discrimination and calibration for all-cause mortality was offered by the ACEF CG at 30days, the ACEF MDRD at 1year and similarly by the ACEF CG and ACEF MDRD at 5years. At 30days, the NRI was +32.9% for ACEF MDRD over ACEF SrCr and +16% for ACEF CG over ACEF SrCr . At 1year, the NRI was 13.8% for ACEF MDRD over ACEF SrCr and −7.8% for ACEF CG over ACEF SrCr . At 5years, the NRI was +7.7% for both the ACEF MDRD and the ACEF CG over the ACEF SrCr . Conclusions In patients undergoing PCI, the ACEF score is associated with satisfactory early-, mid- and long-term discrimination regardless of the definition of renal function. However, incorporating glomerular filtration rate or creatinine clearance by the MDRD or CG formulas in the ACEF score yields superior calibration compared with the original SrCr-based equation, with the ACEF MDRD displaying superior reclassification ability over the ACEF CG and ACEF SrCr at 30days and 1year.

We have constructed a series of MLV-based retroviral vectors and packaging components expressed from the CMV promoter and carried on plasmids containing SV40 origins of replication. These two features greatly enhanced retroviral gene expression when introduced into cell lines carrying the SV40 large T antigen. The two packaging components, gag-pol and env, were placed on separate plasmids to reduce helper virus formation. Using a highly transfectable human cell line and sodium butyrate to further increase expression of each component, we achieved helper-free viral stocks of approximately 10(7) infectious units/ml by 48 h after transient co-transfection with the three plasmid components. This system can be used both for the generation of high titer retroviral stocks for transduction and for the rapid screening of a large number of MLV gag-pol or env mutants.

Cdk9 and Cdk7 are cdc2-like serine/threonine kinases that stabilize RNA transcript elongation through RNA polII carboxyl terminal domain (CTD) phosphorylation and are considered suitable targets for cancer therapy. The effects of flavopiridol and of siRNA-mediated inhibition of Cdk9 and/or Cdk7 were analyzed in human glioblastoma and human prostate cancer cell lines. One finding revealed that Cdk9 and Cdk7 could substitute each other in RNA polII CTD phosphorylation in contrast to the in vitro system. Thus, a simultaneous inhibition of Cdk9 and Cdk7 might be required both for targeting malignant cells and developing a platform for microarray analysis. However, these two pathways are not redundant, as indicated by differential effects observed in cell cycle regulation following siRNA-mediated inhibition of Cdk9 and/or Cdk7 in human PC3 prostate cancer cell line. Specifically, siRNA-mediated inhibition of Cdk9 caused a shift from G 0/G 1 to G 2/M phase in human PC3 prostate cancer cell line. Another finding showed that flavopiridol treatment induced a substantial AKT-Ser473 phosphorylation in human glioblastoma T98G cell line in contrast to siRNA-mediated inhibition of Cdk9 and Cdk9 combined with Cdk7, whereas siRNA-mediated silencing of Cdk7 caused a minor increase in AKT-Ser473 phosphorylation. AKT-Ser473 is a hallmark of AKT pathway activation and may protect cells from apoptosis. This finding also shows that Cdk9 and Cdk7 pathways are not redundant and may have important implications in drug development and for studying the mechanism of chemoresistance in malignant cells.

The focus of the seventh annual meeting of the International Society for Stem Cell Research was placed on several topics, such as cancer stem cells, stem cell transplantation, tissue regeneration, biology of embryonic and adult stem cells of various species and production of induced pluripotent stem cells via reprogramming of mammalian somatic cells. This report provides an overview of stem cell research, based on the proceedings of the meeting.

Recent findings showed certain specific factors had the ability to reprogram in vitro human somatic cells, which in turn could generate pluripotent stem cells. Remarkably, somatic cell-derived pluripotent stem cells recapitulated the features of human embryonic stem cells, as demonstrated independently by two groups of investigators. The reprogramming of somatic cells may represent an alternative to the controversial use of human embryonic stem cells in transplantation therapy and in a variety of other applications, such as generation of cellular models for human diseases and for drug research and development. However, the intrinsic expression of epigenetic reprogramming factors in somatic cells requires the use either of lentiviral- or retroviral-mediated gene transfer, which may cause insertional mutagenesis-induced malignancies. In summary, the recent achievements for the in vitro reprogramming of human somatic cells to a pluripotent state can be considered an important advancement in stem cell research. As for future clinical applications in regenerative medicine, it is necessary to tackle the issue of insertional mutagenesis, which is associated with all types of integrating gene delivery systems. Another critical issue is the control of the tumorigenic potential of somatic cell-derived pluripotent stem cells. As it stands, the newly developed technology can be used to engineer useful models for the study of human diseases and development of novel therapeutics.

Cyclin-dependent kinase 9 (Cdk9) is a cdc2-like serine/threonine kinase. The so-called Cdk9-related pathway comprises two Cdk9 isoforms (Cdk9-42 and Cdk9-55), cyclin T1, cyclin T2a, cyclin T2b and cyclin K. The association between Cdk9 and one of its cyclin partners forms a heterodimer, which is the main component of the positive transcription elongation factor (P-TEFb). The latter stabilizes the elongation process of RNA polymerase II (polII) transcripts. Through the control of RNA polII-mediated gene expression, the Cdk9-related pathway performs an important role in several biological processes, such as cell growth, proliferation, protection from apoptosis and differentiation. Incidentally, the P-TEFb that contains the heterodimer Cdk9-cyclin T1 is also critical for HIV-1 and HIV-2 replication in human cells. A deregulation in the Cdk9-related pathway is associated with various types of human malignancies and cardiomyocytes hypertrophy. On these grounds, the characterization of Cdk9-related pathway deregulation might have a two-fold purpose: (1) the development of novel kinase inhibitors for the treatment of cancer, AIDS and cardiac hypertrophy and (2) a better understanding of the pathogenesis and progression of these maladies.

Tumor suppressor pRb/p105, pRb/p107, and pRb2/p130 genes belong to the retinoblastoma (Rb) gene family. The members of the Rb gene family and the transcription factor E2F play an essential role in regulating cell cycle and, consequently, cell proliferation. This mini-review describes the mechanisms by which Rb family members and E2F regulate cell cycle progression.

Tumor suppressor pRb2/p130 gene belongs to the retinoblastoma (Rb) gene family, which also includes pRb/p105 and pRb/p107. The members of the Rb gene family have attracted a great deal of interest because of their essential role in regulating cell cycle and, consequently, cell proliferation. This mini review discusses the potential therapeutic applications both of pRb2/p130 and its derived product Spa310 spacer domain in cancer treatment. J. Cell. Physiol. 213: 403–406, 2007. © 2007 Wiley-Liss, Inc.

The hematopoietic system consists of: (1) a network of stem and progenitor cells of varying degrees of maturity interacting with other cells that possess supportive and regulatory capacities and (2) vascular stem cell niches with supporting stem cells in their self-renewal, proliferation, differentiation, and mobilization to the circulation. Recent data suggest that selective expression of organ-specific chemokines promotes the mobilization of bone-marrow-derived pluripotent cells, a process that is essential for tissue vascularization and organ regeneration. Despite intensive investigation, the pathways by which mechanical signals are converted to biochemical responses are not completely understood. Recent studies have suggested that chromatin shifts and cell cycle effects stem cell gene expression, and thus results in changes of its surface receptor expression at different points of the cell cycle machinery, therefore changing cell cycle transit. This review will attempt to discuss new approaches to determine the regulation of stem cell growth and differentiation by underlying the significance of the chaotic dynamics of transcriptional networks within a cell, with a combination of chemokines and cytokines in the environment, and mechanical forces, such as; stretch, strain and laminar flow, all involving both cooperation and competition. J. Cell. Physiol. 213:672–678. © 2007 Wiley-Liss, Inc.

Nonviral-mediated gene transfer was used in human gene therapy clinical trials that dealt with the treatment of inherited or acquired genetic disorders and cancer. Several preclinical studies are currently ongoing to employ nonviral vectors in genetic immunization programs for a variety of infectious diseases. The interest in nonviral-mediated gene transfer is motivated by two main reasons: (I) nonviral-based vectors do not derive from infectious agents and are minimally toxic; and (II) they can be easily produced in large quantities. However, the main drawbacks of nonviral-mediated gene transfer are related to low transfection efficiency of target cells, especially in vivo, and to the transient nature of transgene expression. These drawbacks render nonviral-mediated gene transfer not particularly suitable for the treatment of pathological conditions that require long-term transgene expression, such as neurodegenerative disorders and inherited or acquired genetic diseases. On these grounds, the optimal application of nonviral-mediated gene transfer is in immunotherapy for cancer and infectious diseases, as a transient expression of the transgene might be sufficient to trigger effective and durable host immune responses. The purpose of this review is to summarize the standpoint of nonviral vector development.

The transcriptional and chromatin profile of the promoter, first exon and first intron of the human TH gene were analyzed in human neuroblastoma BE(2)-C-16 and human renal carcinoma 293FT cell lines. The latter is a cell culture system that is not permissive for TH gene expression, whereas the former has a 50% cell fraction that tests positive for TH. The engineering of a 6.3 kb recombinant human TH promoter revealed the presence of repressors of transcription between positions (-6,244/-194). The addition of a 1.2 kb fragment of the first intron of the human TH gene (+730/+1,653) enhanced transcriptional activity of the recombinant promoter. However, both constructs were not specific for TH-positive BE(2)-C-16 cells. Chromatin immunoprecipitation (Chip) analysis was carried out on BE(2)-C-16 and 293FT cells to probe sequences of promoter, first exon and first intron of the human TH gene from position (-448/+1,204). The presence of nucleosomes was observed approximately from position (-20/+473) in both cell lines. Chip analysis was then conducted to determine the acetylation of various lysine residues of H3 and H4 in both cell lines. All analyzed lysine residues of H3 and H4 were acetylated in BE(2)-C-16 cells, whereas 293FT cells tested positive for acetylation only in the external lysine residues of the histone tail. Our data are compatible with an active TH gene expression in a 50% cell fraction of BE(2)-C-16 cells. Further analysis of epigenetic programming might lead to the identification of the factors that determine TH gene expression specifically in dopaminergic neurons.

Stem cell research has greatly contributed to the field of oncology with the identification and isolation of cancer stem cells from a variety of tumors. The discovery of rare subpopulations of cancer stem cells has indeed entirely changed the focus of cancer research. Normal adult stem cells and cancer stem cells can both self-renew and undergo a differentiation program that, in turn, gives rise to a high number of differentiated cells. Adult stem cells and their malignant counterparts share almost all of the same intrinsic and extrinsic factors to regulate self-renewal, differentiation and proliferation pathways. Fractions of normal and cancer stem cells are naturally more resistant to toxic injuries than any other cell type. Overall, these observations lead to the conclusion that adult stem or progenitor cells can eventually become malignant by generating cancer stem cells, which are responsible for the development and maintenance of the tumor mass. In addition, chemo-resistant cancer stem cells may cause the relapse of the disease following an apparent beneficial treatment. Indeed, the study of the biology of cancer stem cells might lead to the improvement of preventive cancer diagnosis and to the development of novel therapeutics, which must be designed to selectively target malignant stem cells without affecting normal adult stem cells.

The high immunogenicity of recombinant adenoviral vectors is one of the major issues in the field of gene therapy. Adenoviral-based vectors are susceptible both to cytotoxic T-lymphocyte and humoral immune responses. In addition, leaky adenoviral genes also render transduced cells susceptible to host immune responses. These are the main reasons why adenoviral-based vectors are not suitable to correct genetic disorders, which require long-term expression of the transgene. Another limit to long-term transgene expression is posed by the fact that adenoviral-based vectors do not integrate their genome into the cellular chromosomal DNA of transduced cell populations. As it stands, adenoviral-mediated gene transfer is a promising tool for cancer therapy and for genetic immunization programs against infectious diseases, provided that host immune responses are carefully controlled.

The role of adult stem cells in tissue regeneration has attracted much interest because of its enormous therapeutic potential. Adult stem cells reside in every tissue of the body and have the ability to self-renew and to give rise to a high number of differentiated progeny. These are indeed the essential steps in tissue regeneration, which in some cases occurs constitutively, whereas in other cases happens in response to an injury. The identification of the key factors involved in self-renewal and differentiation pathways is at the top of the agenda of stem cell biologists. Interestingly, a number of factors that govern the fate of adult stem cells also play a role in malignant cell transformation, such as the Wnt cascade, Oct-4, Bmi-1 and Evi1. In addition, rare subpopulations of cancer stem cells were identified for leukemia and other solid tumors. These rare fractions of cancer stem cells are in large part responsible for maintaining the tumor mass, as they have the capacity to self-renew and to generate a high number of progeny via differentiation program. These findings can be considered a major breakthrough in cancer research. Currently, oncologists and stem cell biologists have two main goals: i) the assessment of the consistency of the line that divides adult stem cells and carcinogenesis; ii) to better characterize the biology of cancer stem cells. This new line of investigation may allow for the development of novel therapeutics and cancer diagnostics.

A previous study on the human tyrosine hydroxylase (TH) promoter revealed remarkable differences in the mechanism of TH gene regulation between the human and murine models. Indeed, a low degree of homology was observed in the sequence of TH promoters among human, mouse, and rat systems. Only five short conserved regions (CRs) could be identified among the three species. A human TH minimal promoter was engineered and assembled into a self-inactivating lentiviral vector system. This human TH minimal promoter contained the five CRs plus the first -194 bp from the transcription start of the human TH promoter and the first 35 bp of the untranslated messenger RNA leader of the human TH gene. A significant degree of specificity for this human TH minimal promoter was observed only for human neuronal progenitor cells (hNPCs), but not for TH-positive differentiated mouse primary striatal and substantia nigra cells, indicating a significant difference in TH gene regulation between the human and mouse systems. Not only is the degree of homology between the human and mouse promoters in the range of only 46%, but also those few elements that share a high degree of homology display totally different functions in human and mouse brain-derived cells. In the rodent system, NR4A2 (Nurr1) is required for the transactivation of TH minimal promoters. Intriguingly, neither the dimeric nor the heterodimeric binding sites for Nurr1 are present in the 13 kb DNA sequence that contains the human TH promoter. Instead, the CRs termed one and four of the human TH promoter encode only for a half palindromic binding site sequence for Nurr1, which failed to bind Nurr1 in an in vitro electrophoretic mobility shift assay (EMSA). Additionally, of the three monomeric NGFI-B response element (NBRE) core sites (AGGTCA) and two NBRE-related sites present in the human TH promoter, only one core and two NBRE-related sites formed protein binding complexes. Interestingly, there was no increase of protein binding complex formation upon TH induction and in no case could antibodies supershift Nurr1 from the complex. These findings, taken together, demonstrate that NBRE-related binding sites for Nurr1 do not play a direct role in mediating an interaction between Nurr1 and the human TH promoter. Likewise, immunohistochemical and Western blot analysis have also confirmed that both endogenous and exogenous Nurr1 expression does not positively correlate with TH gene expression in hNPCs, in contrast to the mouse model. In addition, real-time PCR analysis revealed that the downregulation of human Nurr1 gene expression mediated by silencing RNA molecules did not affect human TH gene expression in differentiated hNPCs. A better understanding of human TH gene regulation may have important implications both for the development of novel therapeutic approaches and the study of the pathogenesis of a variety of neurological illnesses, including Parkinson's disease, bipolar disorder, and schizophrenia.

Vectors based on adeno-associated virus (AAV) have recently been used in phase I clinical trials for the treatment of neurological disorders, such as Parkinson's and Canavan's diseases. Indeed, AAV-mediated gene transfer is a promising tool for the delivery of therapeutic gene into the central and peripheral nervous systems. AAV-mediated gene transfer was also applied in phase I and phase II clinical trials for the treatment of cystic fibrosis and in phase I clinical trials for the treatment of hemophilia B. Remarkable progress is being reported in the development of AAV-based vectors; however, the design of AAV-derived vectors needs to be improved. As it stands, AAV-mediated gene transfer has a limited capacity in accommodating foreign genes. In addition, some preclinical studies have shown that AAV-derived vectors can cause tumors in animals due to insertional mutagenesis events. This review will discuss perspectives and drawbacks for AAV-based vector systems.

A DNA fragment of about 13 kb containing the human tyrosine hydroxylase (TH) promoter was previously isolated from a genomic DNA library and sequenced. The 11 kb from the transcription start of the human TH promoter was successively joined to the green fluorescent protein (GFP) to generate a transgenic mouse model. High levels of GFP expression could be observed in TH-positive cells of the Substantia nigra of embryonic and adult mice. Intriguingly, the sequence of the human TH promoter showed a low degree of homology with the mouse and rat TH promoters. In fact, comparative analysis of the sequences of human, rat, and mouse TH promoters revealed only five small regions of high homology. These five evolutionarily conserved regions were numbered in numeric progression from the 5' end of human TH promoter. In the present study, a panel of minimal human TH promoters was generated to analyze the transcriptional activity and specificity of gene expression conferred by the five conserved regions (CRs). The series of constructs was termed 250 bp and contained the first -194 bp of the human TH promoter immediately upstream of the transcription start, the first 35 bp the human TH messenger RNA leader, plus one or more of the five CRs. All the constructs were assembled in a self-inactivating form of the latest series of lentiviral vector system based on the human immunodeficiency virus type 1 (HIV-1). Lentiviral-mediated gene transfer was highly efficient for the in vitro transduction of human neuronal progenitor cells (hNPCs). Since a subset of hNPCs express TH following in vitro treatment with a mixture of differentiating agents, it was possible to assess specificity of expression for all the minimal human TH promoters. Overall, the successive addition of the five conserved regions produced a greater degree of specificity in induced TH-positive hNPCs, in particular after the addition of CRI (-8,917, -8,876). However, the human TH minimal promoters did not show any specificity for TH-positive differentiated mouse primary striatal and S. nigra cells, indicating a difference of TH gene regulation between the human and mouse systems. The human TH minimal promoters may provide the opportunity for the selection of TH-positive human embryonic and adult stem cells for brain transplantation experiments in animal models for Parkinson's disease.

The possibility of regenerating tissues would provide an effective therapeutic tool for the treatment of many pathological conditions, including neurological diseases, spinal cord injuries, cardiopathies, diabetes, hematological illnesses and genetic disorders. While stem cells may have the potential of regenerating a variety of tissues, as indicated by a number of groundbreaking but preliminary reports, ethical issues and safety considerations seem to preclude the use of human embryonic stem cells in the clinical setting. Adult stem cells might circumvent the issues posed by embryonic stem cells, although the potential plasticity of adult stem cells is under scrutiny because of many conflicting and contradictory reports in the field of stem cell research. Indeed, many aspects of the biology of stem cells are still not known. In this respect, stem cell biologists have to address several pressing issues. A better understanding of stem cell biology would almost certainly allow for the establishment of efficient and reliable cell transplantation experimental programs in the clinic.

Purpose: In addition to their original function as cell cycle regulators, retinoblastoma (Rb) family members were recently reported to modulate the sensitivity of cancer cells to chemotherapeutic agents. The purpose of this study is to investigate the possible role of pRb2/p130 in the sensitivity of ovarian cancer to camptothecin, doxorubicin, and taxol. Experimental Design: pRb2/p130 was overexpressed in the CAOV-3 ovarian cancer cell line, and the effect of pRb2/p130 overexpression on sensitivity to apoptosis trigged by IC50 doses of different drugs was evaluated by various methods, including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, flow cytometry, and Western blot analyses. Results: The results reported in this study support the conclusion that overexpression of pRb2/p130 in the CAOV-3 ovarian cancer cell line lacking wild-type p53 is able to inhibit apoptosis triggered by camptothecin and doxorubicin through the c-Jun NH2-terminal kinase signaling transduction pathway. Conversely, taxol-induced cell death is not influenced by the pRb2/p130 protein level. Conclusions: A careful analysis of pRb2/p130 expression in tumor specimens could help to identify the best clinical protocol to be used for each patient, improving efficacy and tolerance and therefore offering additional progress in the treatment of advanced ovarian cancer.