Loss Of Proliferation Potential Research Articles

Functional characterization and genomic studies of a novel murine submandibular gland epithelial cell line.

A better understanding of the normal and diseased biology of salivary glands (SG) has been hampered, in part, due to difficulties in cultivating and maintaining salivary epithelial cells. Towards this end, we have generated a mouse salivary gland epithelial cell (mSGc) culture system that is well-suited for the molecular characterization of SG cells and their differentiation program. We demonstrate that mSGc can be maintained for multiple passages without a loss of proliferation potential, readily form 3D-spheroids and importantly express a panel of well-established salivary gland epithelial cell markers. Moreover, mSGc 3D-spheroids also exhibit functional maturation as evident by robust agonist-induced intracellular calcium signaling. Finally, transcriptomic characterization of mSGc by RNA-seq and hierarchical clustering analysis with adult organ RNA-seq datasets reveal that mSGc retain most of the molecular attributes of adult mouse salivary gland. This well-characterized mouse salivary gland cell line will fill a critical void in the field by offering a valuable resource to examine various mechanistic aspects of mouse salivary gland biology.

Suppression of Sclerostin Alleviates Radiation-Induced Bone Loss by Protecting Bone-Forming Cells and Their Progenitors Through Distinct Mechanisms.

Focal radiotherapy is frequently associated with skeletal damage within the radiation field. Our previous in vitro study showed that activation of Wnt/β-catenin pathway can overcome radiation-induced DNA damage and apoptosis of osteoblastic cells. Neutralization of circulating sclerostin with a monoclonal antibody (Scl-Ab) is an innovative approach for treating osteoporosis by enhancing Wnt/β-catenin signaling in bone. Together with the fact that focal radiation increases sclerostin amount in bone, we sought to determine whether weekly treatment with Scl-Ab would prevent focal radiotherapy-induced osteoporosis in mice. Micro-CT and histomorphometric analyses demonstrated that Scl-Ab blocked trabecular bone structural deterioration after radiation by partially preserving osteoblast number and activity. Consistently, trabecular bone in sclerostin null mice was resistant to radiation via the same mechanism. Scl-Ab accelerated DNA repair in osteoblasts after radiation by reducing the number of γ-H2AX foci, a DNA double-strand break marker, and increasing the amount of Ku70, a DNA repair protein, thus protecting osteoblasts from radiation-induced apoptosis. In osteocytes, apart from using similar DNA repair mechanism to rescue osteocyte apoptosis, Scl-Ab restored the osteocyte canaliculi structure that was otherwise damaged by radiation. Using a lineage tracing approach that labels all mesenchymal lineage cells in the endosteal bone marrow, we demonstrated that radiation damage to mesenchymal progenitors mainly involves shifting their fate to adipocytes and arresting their proliferation ability but not inducing apoptosis, which are different mechanisms from radiation damage to mature bone forming cells. Scl-Ab treatment partially blocked the lineage shift but had no effect on the loss of proliferation potential. Taken together, our studies provide proof-of-principle evidence for a novel use of Scl-Ab as a therapeutic treatment for radiation-induced osteoporosis and establish molecular and cellular mechanisms that support such treatment. © 2016 American Society for Bone and Mineral Research.

Functional and Molecular Changes of MSCs in Aging.

Mesenchymal stem cells (MSCs) are ideal candidates for different cellular therapies due to their simple isolation, extensive expansion potential, and low immunogenicity. For various therapeutic approaches, such as bone and cartilage repair, MSCs are expected to replace the damaged tissues by direct differentiation. However, age-related changes in MSCs lead to the loss of differentiation potential, loss of proliferation potential and increase in senescent cell numbers, which involve a steady loss of bone mass and frequently result in osteoporosis. In this review, we will introduce the characteristic and age-related changes of MSCs. In addition, we will also summarize the potential rescue mechanisms of age-related bone loss involved in differentiation regulation and proliferation regulation, including transcription factors, signal pathways, epigenetic regulation, and oxidative stress regulation.

DNA methylation in stem cell renewal and multipotency

Owing to their potential for differentiation into multiple cell types, multipotent stem cells extracted from many adult tissues are an attractive stem cell resource for the replacement of damaged tissues in regenerative medicine. The requirements for cellular differentiation of an adult stem cell are a loss of proliferation potential and a gain of cell-type identity. These processes could be restricted by epigenetic modifications that prevent the risks of lineage-unrelated gene expression or the undifferentiated features of stem cells in adult somatic cells. In this review, we focus on the role of DNA methylation in controlling the transcriptional activity of genes important for self-renewal, the dynamism of CpG methylation of tissue-specific genes during several differentiation programs, and whether the multilineage potential of adult stem cells could be imposed early in the original precursor stem cells through CpG methylation. Additionally, we draw attention to the role of DNA methylation in adult stem cell differentiation by reviewing the reports on spontaneous differentiation after treatment with demethylating agents and by considering the evidence provided by reprogramming of somatic cells into undifferentiated cells (that is, somatic nuclear transfer or generation of induced pluripotent cells). It is clear from the evidence that DNA methylation is necessary for controlling stem cell proliferation and differentiation, but their exact contribution in each lineage program is still unclear. As a consequence, in a clinical setting, caution should be exerted before employing adult stem cells or their derivatives in regenerative medicine and appropriate tests should be applied to ensure the integrity of the genome and epigenome.

Manganese Superoxide Dismutase Protects the Proliferative Capacity of Confluent Normal Human Fibroblasts

We tested the hypothesis that manganese superoxide dismutase (MnSOD), an antioxidant enzyme, regulates the proliferative potential of confluent human fibroblasts. Normal human skin (AG01522) and lung (WI38, CCL-75) fibroblasts kept in confluence (>95% G(0)/G(1)) showed a significant decrease in their capacity to re-enter the proliferation cycle after 40-60 days. The inhibition of re-entry was accompanied with the age-dependent increase of p16 protein levels in the confluent culture. Adenoviral mediated overexpression of MnSOD during confluent growth suppressed p16, enhanced p21 protein accumulation, and protected fibroblasts against the loss of proliferation potential. Increases in p21 protein levels in MnSOD overexpressing confluent fibroblasts were independent of p53 protein levels. p53 protein levels did not change in control, replication-defective adenovirus containing an insertless vector (AdBgl II), or AdMnSOD-infected confluent cells cultured for 20 and 60 days. In addition, MnSOD-induced protection of the proliferation capacity of confluent fibroblasts was independent of their telomerase activity. However, telomerase-transformed fibroblasts showed increased MnSOD expression in confluent growth, maintaining their capacity to re-enter the proliferation cycle. Although inactivation of the retinoblastoma protein in cells subcultured from the 60-day confluent control, AdBgl II-, and AdMnSOD-infected fibroblasts was identical, only MnSOD-overexpressing cells showed a higher percentage of S-phase. These results support the hypothesis that a redox-sensitive checkpoint regulated the progression of fibroblasts from G(0)/G(1) to S-phase.

Migrational analysis of the constitutively proliferating subependyma population in adult mouse forebrain

Initial experiments to evaluate the in vivo fate(s) of constitutively proliferating subependymal cells determined that, following in vivo labeling of this population by infection with a retrovirus containing a β-galactosidase reporter gene, there was a progressive and eventually complete loss of histochemically β-galactosidase-positive cells within the lateral ventricle subependyma with increasing survival times of up to 28 days after retroviral infection. Subsequent experiments were designed to ascertain the potential contributions of: (i) the migration of subependymal cells away from the forebrain lateral ventricles; and (ii) the down-regulation of the retroviral reporter gene expression. Retroviral lineage tracing experiments demonstrate that a major in vivo fate for constitutively proliferating subependymal cells is their rostral migration away from the walls of the lateral ventricle to the olfactory bulb. Although down-regulation of retroviral reporter gene expression does not contribute to the loss of detection of β-galactosidase-labeled cells from the lateral ventricle subependyma, it does result in an underestimation of the absolute number of retrovirally labeled cells in the olfactory bulb at longer survival times. Furthermore, a temporal decrease in the double labeling of β-galactosidase-labeled cells with [ 3H]thymidine was observed, indicating that only a subpopulation of the migratory subependymal-derived cells continue to actively proliferate en route to the olfactory bulb. These two events may contribute to the lack of a significant increase in the total number of retrovirally labeled subependymal cells during rostral migration. Evidence from separately published studies suggests that cell death is also an important regulator of the size of the constitutively proliferating subependymal population. In summary, in vivo studies utilizing retroviral reporter gene labeling demonstrate that constitutively proliferating subependymal cells born in the lateral ventricle migrate rostrally to the olfactory bulb. Loss of proliferation potential and retroviral reporter gene down-regulation contribute to the lack of any significant increase in the total number of labeled cells recovered in the olfactory bulb.

TAXOTERE(R) INHIBITS THE PROLIFERATION AND INDUCES THE DIFFERENTIATION OF HL-60 AND K562 HUMAN MYELOID-LEUKEMIA CELLS IN-VITRO

Taxotere(R), a synthetic taxoid, has shown strong antitumor activity in vitro, in animal models and in human ovarian and breast carcinomas and malignant melanoma xenografts. Using clonogenic microassays with agar-containing glass capillaries we confirmed the potent inhibition of Taxotere(R) on the colony formation of murine bone marrow cells (GM-CFC: IC50 = 2.3 x 10(-8) M), human myeloid K562 (IC50 = 4.6 x 10(-9) M), HL-60 leukemia (IC50 = 4.6 x 10(-10) M) and KB human squamous carcinoma cells (IC50 = 2.1 x 10(-9) M). Thus, these tumor cells were 5 to 50 times more sensitive to Taxotere(R) than normal GM-CFU-C. In comparison, Taxotere(R) was found to be 50 to 350 fold more effective on the same tumor cells than cis-platin on the basis of the IC50 values. It is noteworthy that dose-response curves of Taxotere(R) and cis-platin presented different shapes suggesting that Taxotere(R) may have a more favorable therapeutic index. Moreover, Taxotere(R) was also able to induce HL-60 cells to differentiate to monocytes between 10(-10) and 10(-9) M and K562 cells between 10(-9) and 10(-8) M. The differentiation inducing capacity of Taxotere(R) was inversely related to the loss of proliferation potential and occurred at concentrations that did not significantly affect cell viability; hence a direct cytotoxic killing effect may be precluded. Thus, the antiproliferative activity of Taxotere(R) may be attributed not only to its spindle poison like action, but also to its differentiation inducing capacity. These data suggest the need for further studies to evaluate Taxotere(R) in' combination cytotoxic-differentiation therapy.