Upregulated expression of lamin B receptor increases cell proliferation and suppresses genomic instability: implications for cellular immortalization.

Mammalian somatic cells undergo terminal proliferation arrest after a limited number of cell divisions, a phenomenon termed cellular senescence. However, cells acquire the ability to proliferate infinitely (cellular immortalization) through multiple genetic alterations. Inactivation of tumor suppressor genes such as p53, RB and p16 is important for cellular immortalization, although additional molecular alterations are required for cellular immortalization to occur. Here, we aimed to gain insights into these molecular alterations. Given that cellular immortalization is the escape of cells from cellular senescence, genes that regulate cellular senescence are likely to be involved in cellular immortalization. Because senescent cells show altered heterochromatin organization, we investigated the implications of lamin A/C, lamin B1 and lamin B receptor (LBR), which regulate heterochromatin organization, in cellular immortalization. We employed human immortalized cell lines, KMST-6 and SUSM-1, and found that expression of LBR was upregulated upon cellular immortalization and downregulated upon cellular senescence. In addition, knockdown of LBR induced cellular senescence with altered chromatin configuration. Additionally, enforced expression of LBR increased cell proliferation likely through suppression of genome instability in human primary fibroblasts that expressed the simian virus 40 large T antigen (TAg), which inactivates p53 and RB. Furthermore, expression of TAg or knockdown of p53 led to upregulated LBR expression. These observations suggested that expression of LBR might be upregulated to suppress genome instability in TAg-expressing cells, and, consequently, its upregulated expression assisted the proliferation of TAg-expressing cells (i.e. p53/RB-defective cells). Our findings suggest a crucial role for LBR in the process of cellular immortalization.

Extract of Plumbago zeylanica enhances the growth of hair follicle dermal papilla cells with down-regulation of 5α-reductase type II.

BACKGROUND: Cellular senescence, a phenomenon of irreversible growth arrest of mammalian cells, is involved in various age-related phenomena in organisms. Hair follicle dermal papilla (DP) cells play important roles in the regulation of hair growth and loss. AIMS: We examined the implication of cellular senescence of DP cells in androgenetic alopecia (AGA), the most common form of male hair loss, and searched for the compounds that have a beneficial effect on the prevention of AGA. PATIENTS/METHODS: Expression of the 5α-reductase type II (SRD5A2) gene, which plays a key role in the development of AGA, was examined by quantitative RT-PCR and Western blotting analysis in DP cells. Besides, DP cells were cultured with the extracts of herbs used in traditional Ayurvedic medicine to search for the compounds that have a beneficial effect on the growth of DP cells. RESULTS: We found that expression of the SRD5A2 was up-regulated in senescent DP cells. We also found that the herbal extract of Plumbago zeylanica (root) enhanced the growth of DP cells and down-regulated the expression of SRD5A2 in DP cells. Further, plumbagin, an ingredient of P zeylanica, would be responsible for the above effects of P zeylanica. CONCLUSION: These results suggested the possibility that senescent DP cells may have a role in the development of AGA through up-regulating SRD5A2 expression, and the P zeylanica extract and plumbagin may suppress its development through enhancing the growth of DP cells and down-regulating SRD5A2 expression in DP cells.

Extract of Emblica officinalis enhances the growth of human keratinocytes in culture.

OBJECTIVE: Keratinocytes are the predominant cell type in the epidermis and play key roles in epidermal function. Thus, identification of the compounds that regulate the growth of keratinocytes is of importance. Here we searched for such compounds from the herbs used in traditional medicine Ayurveda. METHODS: Human keratinocytes were cultured in the presence or absence of the herbal extracts for 2 weeks; the effect of the extracts on cell growth was determined by staining the cells with Coomassie brilliant blue. To detect the compounds that regulate the growth of keratinocytes, the herbal extracts were subjected to high-performance liquid chromatography (HPLC). RESULTS: We found that the extract of Emblica officinalis enhanced the growth of keratinocytes in culture. Further, we fractionated the extract of E. officinalis using HPLC and identified the fractions responsible for the enhanced growth of keratinocytes. CONCLUSION: The extract of E. officinalis enhanced the growth of human keratinocytes in culture. E. officinalis contains the compounds that would be beneficial for human skin health because enhanced growth of keratinocytes would promote wound healing.

High concentrations of NaCl induce cell swelling leading to senescence in human cells.

Cell swelling and retardation in DNA replication are always observed in senescent cells. When DNA replication is slowed down with RNA and protein syntheses unchanged in proliferating cells, it causes a phenomenon known as unbalanced growth. The purpose of this study is to assess the role of cell swelling in unbalanced growth in terms of senescence and investigate the mechanism underlying this phenomenon. We tried to induce cell swelling with minimum damage to cells in this study. We perturbed the osmoregulatory functions to induce cell swelling under hypotonic and hypertonic conditions in normal human fibroblasts. Addition of excess NaCl was found to induce significant cell and nuclear swelling in dose- and time-dependent manners. Excess NaCl immediately retarded DNA replication, accumulated cells at G1 phase of the cell cycle, and eventually deprived division potential of the cells. Such cells showed typical senescent cell shape followed by expression of the typical senescence-associated genes. Excess NaCl also activated ERK1/2, p38, and JNK of the mitogen activated protein kinase family. Addition of U0126, an inhibitor of ERK1/2, prevented appearance of senescent features induced by excess NaCl. These results suggest that hypertonic conditions induce cell swelling due to unbalanced growth, thereby leading to cellular senescence.

Molecular basis for premature senescence induced by surfactants in normal human cells.

Sublethal doses of surfactants as exemplified by NP-40 clearly induce premature senescence in normal human cells. To understand molecular basis for this phenomenon, we tried to suppress it with use of various inhibitors. An inhibitor of p38 of the MAPK family almost completely suppressed growth arrest and morphological changes induced by surfactants; however, other inhibitors tested had no effect. Oleic acid, a weak inducer of premature senescence, was found to suppress the effect of NP-40. Fluorescein-labeled oleic acid rapidly bound to the cell surface, and this binding was clearly blocked by pre-treatment with surfactants, suggesting that surfactants and oleic acid compete for binding to the cell surface. Moderate concentrations of cycloheximide, an inhibitor of protein synthesis, also suppressed the senescent features induced by NP-40. These results suggest that surfactants activate p38 signaling pathway by binding to the cell surface, and induce cellular senescence.

Sublethal doses of surfactants induce premature senescence in normal human skin cells.

We evaluated the cytotoxicity of surfactants in human cells. Synthetic surfactants showed different cytotoxicity levels depending on their structures. The cytotoxicity of commercial washing products was determined mainly by the contents of surfactants. All of them induced premature senescence in normal cells, but not in tumor-derived or immortalized cells, under sublethal conditions. Residual surfactants might be a risk factor for skin aging.

Coordinate expression of the human pregnancy-specific glycoprotein gene family during induced and replicative senescence.

Pregnancy-specific glycoproteins (PSGs) comprise a family of highly similar polypeptides encoded by 11 transcriptionally active genes that compactly cluster on band 19q13.2. All members of the PSG family were found to be markedly up-regulated by addition of 5-bromodeoxyuridine in HeLa cells. Similarly, all of the members were markedly up-regulated during replicative senescence in normal human fibroblasts. Promoter analysis of the PSG1, 4, and 11 genes in HeLa cells did not reveal a cis-regulatory element responsive to 5-bromodeoxyuridine in their 5'-flanking sequences. These results suggest that the PSG genes are regulated at a level of higher order chromatin structure besides by a signal of pregnancy.