Current regulatory issues in cell and tissue therapy

Cell-based therapies have grown dramatically in power and scope in recent years. Once limited to blood and BM transplantation, these therapies now encompass tissue repair and regeneration, metabolic support, gene replacement, and immune effector functions, with established and investigational clinical applications in disorders affecting nearly every tissue and organ system. The complexity and novel applications of human cells, tissues, and cellular and tissue-based products (HCT/Ps), however, present potential risks for adverse events. The US Food and Drug Administration, responding to these concerns, has established a tiered, risk-based regulatory structure, in which more rigorous controls and safeguards are required for products thought to pose increased risk. The proposed good tissue practices (GTP) rule and existing good manufacturing practices (GMP) requirements form the principal elements of this regulatory framework. The proposed GTPs are intended to prevent HCT/P contamination with infectious disease agents, and to ensure that these cells and tissues maintain their integrity and function. GMPs focus on production of safe, pure, and potent products, and entail a higher level of process control and product characterization. All HCT/Ps will be required to comply with GTPs. HCT/Ps considered to present greater risks of adverse events, however, will be subject to both GTPs and GMPs, and must obtain premarket approval using the Investigational New Drug (IND) mechanism established for biologics. Although these requirements will present significant challenges for clinician- investigators and laboratories producing HCT/Ps, the regulations fundamentally support good clinical care by increasing safety and control, and enable good science by improving the quality and reliability of data.     

Bax-dependent regulation of Bak by voltage-dependent anion channel 2

Many studies have demonstrated a critical role of Bax in mediating apoptosis, but the role of Bak in regulating cancer cell apoptotic sensitivities in the presence or absence of Bax remains incompletely understood. Using isogenic cells with defined genetic deficiencies, here we show that in response to intrinsic, extrinsic, and endoplasmic reticulum stress stimuli, HCT116 cells show clear-cut apoptotic sensitivities in the order of Bax+/Bak+ > Bax+/Bak- > Bax-/Bak+ > Bax-/Bak-. Small interference RNA-mediated knockdown of Bak in Bax-deficient cells renders HCT116 cells completely resistant to apoptosis induction. Surprisingly, however, Bak knockdown in Bax-expressing cells only slightly affects the apoptotic sensitivities. Bak, like Bax, undergoes the N terminus exposure upon apoptotic stimulation in both Bax-expressing and Bax-deficient cells. Gel filtration, chemical cross-linking, and co-immunoprecipitation experiments reveal that different from Bax, which normally exists as monomers in unstimulated cells and is oligomerized by apoptotic stimulation, most Bak in unstimulated HCT116 cells exists in two distinct protein complexes, one of which contains voltage-dependent anion channel (VDAC) 2. During apoptosis, Bak and Bax form both homo- and hetero-oligomeric complexes that still retain some VDAC-2. However, the oligomeric VDAC-2 complexes are diminished, and Bak does not interact with VDAC-2 in Bax-deficient HCT116 cells. These results highlight VDAC-2 as a critical inhibitor of Bak-mediated apoptotic responses.     

Evaluating the Connectivity of a Protected Areas' Network under the Prism of Global Change: The Efficiency of the European Natura 2000 Network for Four Birds of Prey

Climate and land use changes are major threats to biodiversity. To preserve biodiversity, networks of protected areas have been established worldwide, like the Natura 2000 network across the European Union (EU). Currently, this reserve network consists of more than 26000 sites covering more than 17% of EU terrestrial territory. Its efficiency to mitigate the detrimental effects of land use and climate change remains an open research question. Here, we examined the potential current and future geographical ranges of four birds of prey under scenarios of both land use and climate changes. By using graph theory, we examined how the current Natura 2000 network will perform in regard to the conservation of these species. This approach determines the importance of a site in regard to the total network and its connectivity. We found that sites becoming unsuitable due to climate change are not a random sample of the network, but are less connected and contribute less to the overall connectivity than the average site and thus their loss does not disrupt the full network. Hence, the connectivity of the remaining network changed only slightly from present day conditions. Our findings highlight the need to establish species-specific management plans with flexible conservation strategies ensuring protection under potential future range expansions. Aquila pomarina is predicted to disappear from the southern part of its range and to become restricted to northeastern Europe. Gyps fulvus, Aquila chrysaetos, and Neophron percnopterus are predicted to locally lose some suitable sites; hence, some isolated small populations may become extinct. However, their geographical range and metapopulation structure will remain relatively unaffected throughout Europe. These species would benefit more from an improved habitat quality and management of the existing network of protected areas than from increased connectivity or assisted migration.     

A functional link between Polo-like kinase 1 and the mammalian Target-Of-Rapamycin pathway?

Polo like kinase-1 is a key effector of cell division and its over-expression in several cancers is often linked with negative prognostic. We recently described that Plk1 is over-expressed in acute myeloid leukemia, and that its inhibition selectively reduces the proliferation of leukemic cells. Here, we report that Plk1 inhibition or depletion using pharmacological and siRNA approaches decreased the phosphorylation of two mTOR substrates in AML cells. In HCT116 cells, inducible expression of a constitutively active form of Plk1 leads to activation of mTOR, as shown by increased phosphorylation of its 4E-BP1 and RPS6 down-stream targets. In addition, HCT116 cells over-expressing the active form of Plk1 were characterized by abnormal growth that could be reversed by rapamycin, a specific inhibitor of the TORC1 complex. Altogether these data suggest the existence of a molecular and functional link between the Plk1 mitotic kinase and the mTOR pathway. Given the different established functions of Plk1 and mTOR during the cell cycle, we will discuss the possible meaning of this functional relationship.     

TRAIL promotes membrane blebbing, detachment and migration of cells displaying a dysfunctional intrinsic pathway of apoptosis

Recently, tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL/Apo2L) has been shown to be a potential candidate for cancer therapy. TRAIL induces apoptosis in various cancer cells but not in normal tissues. Here we show that HCT116 and SW480 cells with a deficient mitochondrial apoptotic pathway were resistant to TRAIL-induced apoptosis, whereas HCT116 and SW480 cells with a functional mitochondrial apoptotic pathway underwent apoptosis upon exposure to TRAIL. Surprisingly, TRAIL induced phenotypic changes in cells with a dysfunctional mitochondrial apoptotic pathway, including membrane blebbing and a transient loss of adhesion properties to the substratum. Accordingly, TRAIL stimulated the ability of these cells to migrate. This behavior was the consequence of a transient TRAIL-induced ROCK1 cleavage. In addition, we report that Bax-deficient HCT116 cells exposed to TRAIL for a prolonged period lost their sensitivity to TRAIL as a result of downregulation of TRAIL receptor expression, and became resistant to combination of TRAIL and other drugs such as MG-132 and bortezomib. These findings may have important consequences for TRAIL anti-cancer therapy.     

USP22 acts as an oncogene by the activation of BMI-1-mediated INK4a/ARF pathway and Akt pathway

Recent studies provided strong support for the view that ubiquitin-specific protease 22 (USP22) plays a central role in cell-cycle progression and also in pathological processes such as oncogenesis. We have recently shown that USP22 levels are elevated in colorectal carcinoma with associated increase in the expression of several cell-cycle-related genes. However, the precise mechanism for these functions of USP22 at molecular level has not been fully elucidated. Currently, we investigated the role of USP22 in human colorectal cancer (CRC). We observed that USP22 expression was statistically significantly correlated positively with that of BMI-1, c-Myc and both, pAkt (Ser473), and pAkt (Thr308), in primary tumor tissues from 43 CRC patients. Down-regulation of USP22 expression in HCT116 colorectal cancer cells by siRNA resulted in the accumulation of cells in the G1 phase of the cell cycle. RNAi-knockdown of USP22 in HCT16 cells also led to the repression of BMI-1 and was accompanied by the up-regulation of p16INK4a and p14ARF, with a consequent decrease in E2F1 and p53 levels. In addition, down-regulation of c-Myc-targeted cyclin D2 was also noticed in cells treated with USP22-siRNA. Furthermore, our results showed that USP22 deletion also caused down-regulation of Akt/GSK3β activity, which can also contribute to the reduction of cyclin D2. Collectively, our current results suggest that USP22 may act as an oncogene in CRC as it positively regulates cell cycle via both BMI-1-mediated INK4a/ARF pathway and Akt signaling pathway.     

The cationic cell-penetrating KT2 peptide promotes cell membrane defects and apoptosis with autophagy inhibition in human HCT 116 colon cancer cells

The anticancer activity of cationic antimicrobial peptides (AMPs) has become more interesting because some AMPs have selective recognition against cancer cells. However, their antitumor properties and underlying mechanisms in cancer cells have not been clearly understood. In this study, we evaluated the effects of KT2 (lysine/tryptophan-rich AMP) on the cellular uptake and internalization mechanism, cell viability, surface charge of the cell membrane, membrane integrity, apoptotic cell death, and autophagy in human HCT 116 colon cancer cells. We found that KT2 interacted with the cell membrane of HCT 116 cells and was internalized into HCT 116 cells via clathrin-mediated and caveolae-mediated endocytosis mechanisms. The interaction of KT2 with cells caused cell membrane structure change, elevated membrane permeability, and KT2 also affected the lipid component. The results of atomic force microscopy showed cellular membrane defects of KT2-treated cells. The internalized KT2 induced nuclear condensation and apoptotic cell death. It elevated the apoptotic factor levels including those of cytochrome c and apoptosis-inducing factor. Furthermore, KT2 inhibited autophagy by the suppression of autophagy-related 5, autophagy-related 7, autophagy-related 16 like 1, and Beclin-1 proteins. In conclusion, these results revealed the cytotoxicity of cationic KT2 against HCT 116 cells and may help to clarify the interactions between cationic AMPs and cancer cells.     

Characterization of dihydroartemisinin-resistant colon carcinoma HCT116/R cell line

Dihydroartemisinin (DHA) is an important artemisinin derivative and presents profound anti-tumor potential. A DHA-resistant cell line named HCT116/R derived from colon carcinoma cell line HCT116 was established in our previous study. Herein, we found that HCT116/R cells were much more resistant to DHA- or artesunate-induced proliferation inhibition and more tolerant to DHA-induced cell cycle arrest and apoptosis compared with those of the parent HCT116 cells. The protein levels of P-glycoprotein and MDR-associated protein 1 and the accumulation of doxorubicin in cells were similar in both cell lines. Moreover, HCT116/R cells were still sensitive to camptothecin- and doxorubicin-induced cell growth inhibition. To further explore the characterization of HCT116/R cell line, a proteomic study employing two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry was performed. Eight different expressed proteins between the two cell lines were identified including some heat shock proteins, annexins, etc. This study not only indicates that exposure to DHA may not induce a tumor multi-drug-resistant phenotype but also affords new clues for the further investigation of the anti-cancer mechanisms of DHA and other artemisinin derivatives.     

Intra-Tumoral Heterogeneity in Metastatic Potential and Survival Signaling between Iso-Clonal HCT116 and HCT116b Human Colon Carcinoma Cell Lines

Background

Colorectal cancer (CRC) metastasis is a leading cause of cancer-related deaths in the United States. The molecular mechanisms underlying this complex, multi-step pathway are yet to be completely elucidated. Recent reports have stressed the importance of intra-tumoral heterogeneity in the development of a metastatic phenotype. The purpose of this study was to characterize the intra-tumoral phenotypic heterogeneity between two iso-clonal human colon cancer sublines HCT116 and HCT116b on their ability to undergo metastatic colonization and survive under growth factor deprivation stress (GFDS).

Materials and Methods

HCT116 and HCT116b cells were transfected with green fluorescence protein and subcutaneously injected into BALB/c nude male mice. Once xenografts were established, they were excised and orthotopically implanted into other male BALB/c nude mice using microsurgical techniques. Animal tissues were studied for metastases using histochemical techniques. Microarray analysis was performed to generate gene signatures associated with each subline. In vitro assessment of growth factor signaling pathway was performed under GFDS for 3 and 5 days.

Results

Both HCT116 and HCT116b iso-clonal variants demonstrated 100% primary tumor growth, invasion and peritoneal spread. However, HCT116 was highly metastatic with 68% metastasis observed in liver and/or lungs compared to 4% in HCT116b. Microarray analysis revealed an upregulation of survival and metastatic genes in HCT116 cells compared to HCT116b cells. In vitro analysis showed that HCT116 upregulated survival and migratory signaling proteins and downregulated apoptotic agents under GFDS. However, HCT116b cells effectively showed the opposite response under stress inducing cell death.

Conclusions

We demonstrate the importance of clonal variation in determining metastatic potential of colorectal cancer cells using the HCT116/HCT116b iso-clonal variants in an orthotopic metastatic mouse model. Determination of clonal heterogeneity in patient tumors can serve as useful tools to identify clinically relevant biomarkers for diagnostic and therapeutic assessment of metastatic colorectal cancer.     

TNFR2 signaling modulates immunity after allogeneic hematopoietic cell transplantation

Tumor necrosis factor-α (TNF-α) signaling through TNF receptor 2 (TNFR2) plays a complex immune regulatory role in allogeneic hematopoietic cell transplantation (HCT). TNF-α is rapidly released in the circulation after the conditioning regimen with chemotherapy and/or radiotherapy. It activates the function of donor alloreactive T cells and donor Natural Killer cells and promotes graft versus tumor effects. However, donor alloreactive T cells also attack host tissues and cause graft versus host disease (GVHD), a life-threatening complication of HCT. Indeed, anti-TNF-α therapy has been used to treat steroid-refractory GVHD. Recent studies have highlighted another role for TNFR2 signaling, as it enhances the function of immune cells with suppressive properties, in particular CD4⁺Foxp3⁺ regulatory T cells (Tregs). Various clinical trials are employing Treg-based treatments to prevent or treat GVHD. The present review will discuss the effects of TNFR2 signaling in the setting of allogeneic HCT, the implications for the use of anti-TNF-α therapy to treat GVHD and the clinical perspectives of strategies that specifically target this pathway.     

Business oriented EU human cell and tissue product legislation will adversely impact Member States’ health care systems

The transplantation of conventional human cell and tissue grafts, such as heart valve replacements and skin for severely burnt patients, has saved many lives over the last decades. The late eighties saw the emergence of tissue engineering with the focus on the development of biological substitutes that restore or improve tissue function. In the nineties, at the height of the tissue engineering hype, industry incited policymakers to create a European regulatory environment, which would facilitate the emergence of a strong single market for tissue engineered products and their starting materials (human cells and tissues). In this paper we analyze the elaboration process of this new European Union (EU) human cell and tissue product regulatory regime—i.e. the EU Cell and Tissue Directives (EUCTDs) and the Advanced Therapy Medicinal Product (ATMP) Regulation and evaluate its impact on Member States’ health care systems. We demonstrate that the successful lobbying on key areas of regulatory and policy processes by industry, in congruence with Europe’s risk aversion and urge to promote growth and jobs, led to excessively business oriented legislation. Expensive industry oriented requirements were introduced and contentious social and ethical issues were excluded. We found indications that this new EU safety and health legislation will adversely impact Member States’ health care systems; since 30 December 2012 (the end of the ATMP transitional period) there is a clear threat to the sustainability of some lifesaving and established ATMPs that were provided by public health institutions and small and medium-sized enterprises under the frame of the EUCTDs. In the light of the current economic crisis it is not clear how social security systems will cope with the inflation of costs associated with this new regulatory regime and how priorities will be set with regard to reimbursement decisions. We argue that the ATMP Regulation should urgently be revised to focus on delivering affordable therapies to all who are in need of them and this without necessarily going to the market. The most rapid and elegant way to achieve this would be for the European Commission to publish an interpretative document on “placing on the market of ATMPs,” which keeps tailor-made and niche ATMPs outside of the scope of the medicinal product regulation.     

Inactivation of p21WAF1 sensitizes cells to apoptosis via an increase of both p14ARF and p53 levels and an alteration of the Bax/Bcl-2 ratio

p21(WAF1) appears to be a major determinant of the cell fate in response to anticancer therapy. It was shown previously that HCT116 human colon cancer cells growing in vitro enter a stable arrest upon DNA damage, whereas cells with a defective p21(WAF1) response undergo apoptosis. Here we report that the enhanced sensitivity of HCT116/p21(-/-) cells to chemotherapeutic drug-induced apoptosis correlates with an increased expression of p53 and a modification of their Bax/Bcl-2 ratio in favor of the pro-apoptotic protein Bax. Treatment of HCT116/p21(-/-) cells with daunomycin resulted in a reduction of the mitochondrial membrane potential and in activation of caspase-9, whereas no such changes were observed in HCT116/p21(+/+) cells, providing evidence that p21(WAF1) exerts an antagonistic effect on the mitochondrial pathway of apoptosis. Moreover, the role of p53 in activation of this pathway was demonstrated by the fact that inhibition of p53 activity by pifithrin-alpha reduced the sensitivity of HCT116/p21(-/-) cells to daunomycin-induced apoptosis and restored a Bax/Bcl-2 ratio similar to that observed in HCT116p21(+/+) cells. Enhancement of p53 expression after disruption of p21(WAF1) resulted from a stabilization of p53, which correlated with an increased expression of the tumor suppressor p14(ARF), an inhibitor of the ubiquitin ligase activity of Mdm2. In accordance with the role of p14(ARF) in p53 stabilization, overexpression of p14(ARF) in HCT116/p21(+/+) cells resulted in a strong increase in p53 activity. Our results identify a novel mechanism for the anti-apoptotic effect of p21(WAF1) consisting in maintenance of mitochondrial homeostasis that occurs in consequence of a negative control of p14(ARF) expression.     

Diabetes mellitus: a potential target for stem cell therapy

Type 1 diabetes mellitus has received much attention recently as a potential target for the emerging science of stem cell medicine. In this autoimmune disease, the insulin-secreting beta-cells of the pancreas are selectively and irreversibly destroyed by autoimmune assault. Advances in islet transplantation procedures now mean that patients with the disease can be cured by transplantation of primary human islets of Langerhans. A major drawback in this therapy is the availability of donor islets, and the search for substitute transplant tissues has intensified in the last few years. This review will describe the essential requirements of a material designed as a replacement beta-cell and will look at the potential sources of such replacements. These include embryonic stem (ES) cells and multipotent adult stem/progenitor cells from a range of tissues including the pancreas, intestine, liver, bone marrow and brain. These stem cell populations will be evaluated and the different experimental approaches that have been employed to derive functional insulin-expressing cells will be discussed. The review will also look at the capability of human ES (hES) cells generated by somatic cell nuclear transfer and some adult stem cell populations such as bone marrow-derived stem cells, to offer autologous transplant material that would remove the need for immunosuppression. In patients with Type 1 diabetes, auto-reactive T-cells are programmed to recognise the insulin-producing beta-cells. As a result, for therapeutic replacement tissues, it may be more sensible to derive cells that behave like beta-cells but are immunologically distinct. Thus, the potential of cells derived from non-beta-cell origin to avoid the autoimmune response will also be discussed. Finally, the review will summarise the future prospects for stem cell therapies for diabetes and will highlight some of the problems that may be faced by researchers working in this area, such as malignancy, irreproducible differentiation strategies, immune-system rejection and social and ethical concerns over the use of hES cells.     

Pim-2 phosphorylation of p21Cip1/WAF1 enhances its stability and inhibits cell proliferation in HCT116 cells

Pim-2 kinase is one of the three highly conserved Pim family members which are known to be involved in cell survival and cell proliferation. Here we demonstrate that like Pim-1, Pim-2 also phosphorylates the cell cycle inhibitor p21^Cip1/WAF1 (p21) on Thr145 in vitro and in vivo. Overexpression of Pim-2 in HCT116 cells leads to the increased stability of p21 and results in enhanced levels of both exogenous and endogenous p21 proteins. Knockdown of Pim-2 expression via siRNA results in reduced level of endogenous p21, indicating that like Pim-1, Pim-2 is another legitimate p21 kinase. However, Pim-2 has no influence on the nuclear localization of p21 in HCT116 cells. In addition, Pim-2 is able to arrest the cell cycle at G1/S phase and inhibit cell proliferation through phosphorylation of p21 in HCT116 cells. These data suggest that Pim-2 phosphorylation of p21 enhances p21's stability and inhibits cell proliferation in HCT116 cells.     

PXR基因外显子3甲基化与肠癌细胞对5氟尿嘧啶的耐药性相关

孕烷X受体（pregnane X receptor, PXR）可通过调节细胞色素P450同工酶3A4 -CYP3A4的表达而影响肿瘤细胞对化疗的敏感性,而其表达水平则会受到自身基因 甲基化的影响.本文研究了结肠癌组织中pxr基因甲基化的分布情况及其对pxr, cyp3a4表达的影响,并在多种结肠癌细胞系中分析了pxr基因甲基化是否与5-氟尿嘧 啶 (5-FU)耐药性相关.收集结肠癌病灶区、癌旁区及正常结肠组织样本,分别提取基因组DNA及RNA.PCR限制性酶切分析检测pxr基因外显子3甲基化;real-time PCR检测pxr及cyp3a4基因的表达.鉴定LOVO、LS180、LS174T、HT29、HCT116等5种结 肠癌细胞中pxr外显子3甲基化与pxr, cyp3a4表达的相关性并分别筛选出PXR高/低表达的细胞株进行5-FU耐药性分析.结果显示,结肠癌病灶组织中pxr外显子3甲基化频率显著增加,伴有pxr,cyp3a4表达的增强.在结肠组织及结肠癌细胞系中,pxr与cyp3a4的表达均密切相关,且均与pxr甲基化程度相关.PXR高表达细胞株LS180对5-FU的耐药性显著升高,以siRNA分别下调pxr及cyp3a4的表达,均可增加LS180对5 -FU的敏感性.结果提示,pxr基因外显子3区甲基化与PXR及CYP3A4的高表达密切相关,并与结肠癌细胞对5-FU的抗药性相关.     

The action of Pdcd4 may be cell type specific: evidence that reduction of dUTPase levels might contribute to its tumor suppressor activity in Bon-1 cells

Pdcd4 (programmed cell death protein 4) was identified as a gene up-regulated during apoptosis and, additionally, seems to have a function as a tumor suppressor. However, there are conflicting data concerning its role in programmed cell death and most results for its action as an inhibitor for neoplastic transformation are derived from experiments with epidermal cells. Therefore, we were interested to investigate if the action of Pdcd4 might be cell type specific. For that purpose we examined the expression of Pdcd4 and several other proteins in various tumor cell lines. We could not find any correlation of Pdcd4 levels and expression of proteins associated with cell cycle and/or apoptosis in different cell lines. Furthermore, we stably transfected two cell lines (Bon-1 and HCT116) to over-express Pdcd4 and analyzed protein expression. Although we found several regulated proteins none of these proteins were affected in both cell lines in the same manner. For instance, dUTPase expression was reduced in Bon-1 cells but not changed in HCT116 cells. This regulation might be important for the sensitivity of cells to anti-cancer drugs like inhibitors of thymidilate synthase. Therefore, we conclude that the function of Pdcd4 might be cell type specific. A role for Pdcd4 in apoptosis or as a tumor suppressor might be limited to certain cell types.     

Identification of β integrin‐like‐ and fibronectin‐like proteins in the bivalve mollusk Mytilus trossulus

Integrins play a key role in the intermediation and coordination between cells and extracellular matrix components. In this study, we first determined the presence of the β integrin‐like protein and its presumptive ligand, fibronectin‐like protein, during development and in some adult tissues of the bivalve mollusc Mytilus trossulus. We found that β integrin‐like protein expression correlated with the development and differentiation of the digestive system in larvae. Besides the presence of β integrin‐like protein in the digestive epithelial larval cells, this protein was detected in the hemocytes and some adult tissues of M. trossulus. The fibronectin‐like protein was detected firstly at the blastula stage and later, the FN‐LP‐immunoreactive cells were scattered in the trochophore larvae. The fibronectin‐like protein was not expressed in the β integrin‐positive cells of either the veliger stage larvae or the adult mussel tissues and the primary hemocyte cell culture. Despite the β integrin‐ and fibronectin‐like proteins being expressed in different cell types of mussel larvae, we do not exclude the possibility of direct interaction between these two proteins during M. trossulus development or in adult tissues.