RESUMO
Polysialylation of neural cell adhesion molecule (NCAM) in small-cell lung cancer (SCLC) is thought to regulate NCAM-mediated cell-surface interactions, imparting antiadhesive properties to cells. However, SCLC cells in culture demonstrate anchorage-independent growth and spontaneously generate adherent forms. Here, the ability of polySia-NCAM to influence cell proliferation and adherence is unclear. We analyzed live SCLC cell polySia-NCAM expression by flow cytometry, using the novel combination of a polySia antibody-mimetic eGFP-tagged endosialidase and the viability dye DRAQ7. Enrichment for adherence (<30 population doublings) in SCLC cell lines resolved populations with increased (SHP-77 and COR-L279) or negligible (NCI-H69) polysialylation compared with nonadherent parent populations. Adherent forms retained NCAM expression as confirmed by immunofluorescence and immunoblotting. Initial transition to adherence and loss of polysialylation in NCI-H69 was linked to a reduced proliferation rate with no increase in cell death. This reduced proliferation rate was reiterated in vivo as determined by the growth of noninvasive subcutaneous xenografts in mice. Continued selection for enhanced substrate adherence in NCI-H69 (>150 population doublings) resolved cells with stable re-expression of polySia and increased growth rates both in vitro and in vivo. Endoneuraminidase removal of polySia from re-expressing cells showed that rapid adherence to extracellular matrix components was functionally independent of polySia. PolySia expression was not altered when isolated adherent forms underwent enforced cell-cell contact in three-dimensional culture. Coculture of polySia expression variants modulated overall polySia expression profiles indicating an influence of SCLC microcommunity composition independent of substrate adherence potential. We conclude that an obligatory linkage between substrate adherence potential and polySia expression is rejected for SCLC cells. We suggest that a degree of homeostasis operates to regulate polysialylation within heterogeneous cell populations. The findings suggest a new model for SCLC progression while the application of live cell profiling of polysialylation could be used to assess polySia-NCAM-targeted therapies.
Assuntos
Neoplasias Pulmonares/metabolismo , Ácido N-Acetilneuramínico/metabolismo , Moléculas de Adesão de Célula Nervosa/metabolismo , Carcinoma de Pequenas Células do Pulmão/metabolismo , Animais , Adesão Celular , Linhagem Celular Tumoral , Proliferação de Células , Matriz Extracelular/metabolismo , Citometria de Fluxo , Glicosilação , Humanos , Neoplasias Pulmonares/patologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Invasividade Neoplásica , Transplante de Neoplasias , Processamento de Proteína Pós-Traducional , Carcinoma de Pequenas Células do Pulmão/patologiaRESUMO
Drug resistant tumor "side-populations," enriched in cancer stem cells and identified by reduced accumulation of Hoechst 33342 under ABCG2-mediated efflux, may compromise therapeutic outcome. Side-population cells have predicted resistance to minor groove ligands, including the DNA topoisomerase I poison topotecan. We have used a stable Hoechst 33342-resistant murine L cell system (HoeR415) to study resistance patterns, removing the need for SP isolation before microarray analysis of gene expression and the tracking of cell cycle dynamics and cytotoxicity. The majority of HoeR415 cells displayed a side-population phenotype comparable with that of the side-population resident in the ABCG2 over-expressing A549 lung cancer cell line. Photo-crosslinking showed direct protection against minor groove ligand residence on DNA, driven by ABCG2-mediated efflux and not arising from any binding competition with endogenous polyamines. The covalent minor-groove binding properties of the drug FCE24517 (tallimustine) prevented resistance suggesting a mechanism for overcoming SP-related drug resistance. Hoechst 33342-resistant murine cells showed lower but significant crossresistance to topotecan, again attributable to enhanced ABCG2 expression, enabling cells to evade S-phase arrest. Hoechst 33342/TPT-resistant cells showed limited ancillary gene expression changes that could modify cellular capacity to cope with chronic stress including over-expression of Aldh1a1 and Mgst1, but under-expression of Plk2 and Nnt. There was no evidence to link the putative stem cell marker ALDH1A1 with any augmentation of the TPT resistance phenotype. The study has implications for the patterns of drug resistance arising during tumor repopulation and the basal resistance to minor groove-binding drugs of tumor side-populations.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Benzimidazóis/farmacologia , Citometria de Fluxo/métodos , Topotecan/farmacologia , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Animais , Linhagem Celular Tumoral , Separação Celular , Reagentes de Ligações Cruzadas/farmacologia , Distamicinas/farmacologia , Inibidores Enzimáticos/farmacologia , Corantes Fluorescentes/farmacologia , Humanos , Ligantes , Camundongos , Compostos de Mostarda Nitrogenada/farmacologia , Poliaminas/químicaRESUMO
Metallothioneins (MTs) have an important role in zinc homeostasis and may counteract the impact of oversupply. Both intracellular zinc and MT expression have been implicated in proliferation control and resistance to cellular stress, although the interdependency is unclear. The study addresses the consequences of a steady-state overexpression of MT-1 for intracellular zinc levels, cell cycle progression, and protection from zinc toxicity using a panel of cell lines with differential expression of MT-1. The panel comprised parental Chinese hamster ovary-K1 cells with low endogenous expression of MT and transfectants with enhanced expression of mouse MT-1 on an autonomously replicating expression vector with a noninducible promoter. Cell cycle progression, determined by flow cytometry and time-lapse microscopy, revealed that enhanced cytoplasmic expression of MT-1 does not impact on normal cell cycle operation, suggesting that basal levels of MT-1 expression are not limiting for background levels of oxidative stress. MT-1 overexpression correlated with a steady-state increase in cytoplasmic free Zn(2+), assessed using the fluorescent zinc-sensor Zinquin, particularly at high levels of overexpression, further suggesting that zinc availability is normally not limiting for cell cycle progression. Enhanced MT-1 expression, over a 10-fold range, had a clear impact on resistance to Cd(2+) and Zn(2+) toxicity. In the case of Zn(2+), the degree of protection afforded was less, indicating that MT-1 has a limited range and saturable capacity for effecting resistance. The results have implications for the use of cellular stress responses to exogenously supplied zinc and zinc-based systemic therapies.
Assuntos
Compostos de Cádmio/toxicidade , Ciclo Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Metalotioneína/metabolismo , Estresse Fisiológico/efeitos dos fármacos , Sulfatos/toxicidade , Sulfato de Zinco/toxicidade , Animais , Células CHO , Compostos de Cádmio/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Citoplasma/metabolismo , Citoproteção , Relação Dose-Resposta a Droga , Metalotioneína/genética , Camundongos , Sulfatos/metabolismo , Fatores de Tempo , Transfecção , Regulação para Cima , Sulfato de Zinco/metabolismoRESUMO
In recent years, progress has been made in characterizing the molecular and cellular elements that are responsible for the regeneration in the damaged brain and highlighting the key role of the stromal-vascular 'environment' to orchestrate secondary neurogenesis and repair. Indeed, the ability of the stem cells to self-renew and differentiate is tightly regulated by stromal ependymal cells and endothelial cells expressing molecular cues that constitute the extracellular stem cell 'niche'. Several soluble growth factors such as EGF, TGFbeta, FGF2, SDF-1alpha and Noggin are important signals for the stem cell niche but little is known about the role of membrane-bound molecules in intercellular communications between the niche and the stem cells. In this mini-review, we highlight the emerging role of a family of adhesion molecules in the control of secondary neurogenesis. The coxsackie-adenovirus receptor (CAR) is a 46 kDa transmembrane protein and a member of the immunoglobulin super family. It is close structurally and evolutionary to other adhesion molecules involved in cell-cell interactions during embryogenesis, broadly expressed in the developing central nervous system but restricted to ependymal cells in the adult brain. This unique location and its newly established signalling properties further support the role of CAR in intercellular communications. Elucidating the other signalling molecules and manipulating the stromal-vascular niche for example by adenovirus gene therapy remain important goals for future clinical applications.
Assuntos
Receptores Virais/genética , Receptores Virais/metabolismo , Células-Tronco/fisiologia , Animais , Proteína de Membrana Semelhante a Receptor de Coxsackie e Adenovirus , Humanos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Transdução de Sinais/fisiologiaRESUMO
In invertebrates and primitive vertebrates, the brain contains large numbers of "professional" macrophages associated with neurones, ependymal tanycytes and radial glia to promote robust regenerative capacity. In higher vertebrates, hematogenous cells are largely excluded from the brain, and innate immune molecules and receptors produced by the resident "amateur" macrophages (microglia, astrocytes and ependymal cells) control pathogen infiltration and clearance of toxic cell debris. However, there is minimal capacity for regeneration. The transfer of function from hematogenous cells to macroglia and microglia is associated with the sophistication of a yet poorly-characterized neurone-glia network. This evolutionary pattern may have been necessary to reduce the risk of autoimmune attack while preserving the neuronal web but the ability to repair central nervous system damage may have been sacrificed in the process. We herein argue that it may be possible to re-educate and stimulate the resident phagocytes to promote clearance of pathogens (e.g., Prion), toxic cell debris (e.g., amyloid fibrils and myelin) and apoptotic cells. Moreover, as part of this greater division of labour between cell types in vertebrate brains, it may be possible to harness the newly described properties of glial stem cells in neuronal protection (revitalization) rather than replacement, and to control brain inflammation. We will also highlight the emerging roles of stromal ependymal cells in controlling stem cell production and migration into areas of brain damage. Understanding the mechanisms involved in the nurturing of damaged neurons by protective glial stem cells with the safe clearance of cell debris could lead to remedial strategies for chronic brain diseases.