Over-expression of CDKIs p15INK4b, p16INK4a and p21CIP1/WAF1 genes mediate growth arrest in human osteosarcoma cell lines.

Human somatic cells cultured in vitro exhibit a limited number of divisions. In contrast immortal cells have lost their growth regulatory mechanisms and, thus, continue to divide indefinitely. Cyclin-dependent kinase inhibitors (CDKIs) represent one of the most important regulatory factors in both mortality and immortality, as they are over-expressed in senescent cells and are down-regulated in a number of human cancers. In the present study we determined the effect of CDKIs on the proliferation ability of human osteosarcoma cell lines. Transient expression of various CDKIs (p15INK4b, p16INK4a and p21CIP1/WAF1) in KHOS cells resulted in growth arrest and the cells failed to enter the S-phase of the cell cycle as shown by a DNA synthesis inhibition assay. In addition, stable transfection of p21CIP1/WAF1 and p16INK4a genes in two osteosarcoma cell lines (KHOS and U2-OS cells) showed that p21CIP1/WAF1 was able to repress the immortal phenotype in both cell lines, whereas temporary over-expression of p16INK4a reversibly inhibited the cell growth. Therefore this study indicates that CDKIs mediate growth arrest in human osteosarcoma cell lines and provides further evidence of the existence of molecular links between cellular mortality and immortality.

Cloning and identification of genes that associate with mammalian replicative senescence.

Cellular senescence and limited proliferative capacity of normal diploid cells has a dominant phenotype over immortality of cancerous cells, suggesting its regulation by the expression of a set of genes. In order to isolate the genes that associate with senescence, we have employed a clonal system of conditional SV40 T antigen rat embryo fibroblast cell lines which undergo senescence upon T antigen inactivation. Construction of cDNA libraries from two conditional cell lines and application of differential screening and subtractive hybridization techniques have resulted in the cloning of eight senescence-induced genes (SGP-2/Apo J, alpha 1-procollagen, osteonectin, fibronectin, SM22, cytochrome C oxidase, GTP-alpha, and a novel gene) and a senescence-repressed gene (FRS-2). Three of these genes encode for extracellular matrix proteins, others are involved in the calcium-dependent signal transduction pathways, while the SGP-2/Apo J gene may have a cellular protective function. RNA analysis has shown that the senescence-associated genes are overexpressed in both normal rat embryonic fibroblasts and human osteoblasts cell cultures undergoing aging in vitro. In comparison, the expression of these genes in a rat fibroblast immortalized cell line (208F cells) was down-regulated after both its partial and its full transformation by ras oncogenes. Thus, cloning of senescence-associated genes opens up new ways to elucidate and/or to modulate aging and cancer.

Molecular links between cellular mortality and immortality (review).

Normal diploid cells cultivated in vitro exhibit limited division potential while undergoing ageing during serial passaging. In contrast, cells that have been genetically transformed appear to have lost the regulatory mechanisms of limited growth potential and may continue to divide indefinitely. While cellular mortality is characterised by a progressive cessation of cell growth manifested in cell culture by senescence, immortalisation is the escape from senescence as a result of multiple mechanisms involving the inactivation of tumour suppressor genes, the elevated expression of oncogenes, as well as other genetic and epigenetic changes. The mechanisms governing mortality and immortality are fundamentally linked. The physiological and biochemical features which characterise cellular mortality are examined, herein in the search for markers and timing mechanisms of mortality. The genetic elements involved in the control of mortality and immortality are also discussed, and the fundamental similarities between the molecular and genetic aspects which govern the determination of the phenotypes manifesting the two processes are underlined.

Phorbol ester PMA stimulates protein synthesis and increases the levels of active elongation factors EF-1 alpha and EF-2 in ageing human fibroblasts.

Phorbol esters modulate gene expression, reorganize the cytoskeleton and stimulate bulk protein synthesis and the steps of initiation and elongation. We have observed that a phorbol ester PMA stimulates protein synthesis and increases the amounts of active elongation factors, EF-1 alpha and EF-2 in cultured human fibroblasts MRC-5 undergoing ageing. Although bulk protein synthesis slows down during ageing, the cellular response to the stimulatory effects of PMA is higher in senescent cells. Similarly, despite the age-related decline in the amounts of active EF-1 alpha and EF-2, senescent cells exhibit a higher response to PMA. The results indicate an age-dependent increase of cellular responsiveness to PMA and provide evidence about both the integrity of the translational apparatus and the effectiveness of the signal transduction pathways during cellular ageing. In comparison, the effects of PMA on SV40-transformed MRC-5V2 cells were minimal.

Protein synthesis, posttranslational modifications, and aging.

Posttranslational modifications of proteins are involved in determining their activities, stability, and specificity of interaction. More than 140 major and minor modifications of proteins have been reported. Of these, only a few have been studied in relation to the aging of cells, tissues, and organisms. These include phosphorylation, methylation, ADP-ribosylation, oxidation, glycation, and deamidation. Several of these modifications occur on proteins involved in crucial cellular processes, such as DNA synthesis, protein synthesis, protein degradation, signal transduction, cytoskeletal organization, and the components of extracellular matrix. Some of the modifications are the markers of abnormal and altered proteins for rapid degradation. Others make them less susceptible to degradation by normal proteolytic enzymes, and hence these accumulate during aging.

Senescent human fibroblasts are more sensitive to the effects of a phorbol ester on macromolecular synthesis and growth characteristics.

4-beta-phorbol-12-beta-myristate-13-alpha-acetate (PMA) alters cellular growth properties by modulating gene expression in a wide variety of cell types. Human diploid fibroblasts MRC-5 were treated with PMA at different phases of their lifespan in vitro and the alterations of their short-term growth characteristics and macromolecular synthesis in response to PMA were analysed. PMA stimulates DNA and RNA synthesis in both Phase II (young) and Phase III (senescent) MRC-5 cells. Treatment of senescent cells with various PMA concentrations results in a greater stimulation of DNA and RNA synthesis than that in young cells. Senescent cells are also more sensitive to the PMA-induced alterations of growth characteristics and higher concentrations of PMA become toxic for them. The age-related alterations of cellular responsiveness are also apparent in the gradual loss of responsiveness to serum, observed in parallel with the increased sensitivity to PMA. Furthermore, serum-induced stimulation of macromolecular synthesis is inhibited by PMA. Since it is known that serum and PMA elicit their effects via different signal transduction pathways, our results point to suggest the differential regulation of the signalling mechanisms during cellular ageing.

Structural and histochemical studies of Golgi complex differentiation in salivary gland cells during Drosophila development.

Morphological alterations in the Golgi complex (GC) and changes in the distribution of acid phosphatase (AcPase), thiamine pyrophosphatase (TPPase), complex carbohydrates and reduced osmium tetroxide compounds in this organelle were studied in the salivary gland cells of Drosophila during larval and prepupal development. The morphology and the AcPase, TPPase and complex carbohydrates cytochemical patterns of the Golgi complex varied characteristically during cell differentiation. At the early 3rd instar period the Golgi complex consisted mainly of vesiculated cisternae, and AcPase activity was observed in all cisternae but not in the secretory granules. As development proceeded to the late 3rd instar the Golgi complex displayed its typical appearance, consisting of four to six cisternae, and only the two to three cisternae towards the trans-face as well as the trans-Golgi network and some of the immature secretory granules exhibited AcPase reactivity. In the course of a 'wave' of production of the 'glue' secretory granules proceeding proximally through the gland, the number of AcPase positive cisternae changed correspondingly. After secretion of the 'glue' secretory granules, the size of the Golgi complex decreased and almost all cisternae displayed AcPase reactivity. The detection of TPPase activity presented some specificity problems, since staining was observed not only in the GC cisternae but in the endoplasmic reticulum (ER) and microvilli. The reaction products were seen in a few GC vesicles during the early 3rd instar and in the trans side of the organelle at the end of the 3rd instar. During production of the secretory granules, every GC cisterna was intensely stained. These results agree with previous findings suggesting that AcPase and TPPase in secretory cells may be primarily involved in the processing of exportable proteins. The vicinal (vic)-glycol groups of the complex carbohydrates were detected using the periodic acid/thiocarbohydrazide/silver proteinate (PA-TCH-SP) technique. During synthesis of the 'glue' secretory granules, the reaction products were observed over the GC cisternae and the trans-Golgi network, with increasing intensity from the cis to the trans side of the organelle. No PA-TCH-SP staining was observed over the GC cisternae during the early 3rd instar. Following discharge of the 'glue' secretory granules, all GC cisternae displayed uniform PA-TCH-SP staining. After OsO4 impregnation, the reaction products were observed mainly in ER and mitochondria and rarely in the GC. In numerous cells, only the mitochondria were stained, while in many cases the ER of neighboring cells exhibited differential staining.(ABSTRACT TRUNCATED AT 400 WORDS)

Phorbol ester-induced reorganization of the cytoskeleton in human fibroblasts during ageing in vitro.

Phorbol esters induce drastic morphological alterations in cells of different origin by altering the conformation and the interrelationship of the elements of the cytoskeletal system. Treatment of early passage (young) and late passage (senescent) human fibroblasts MRC-5 with phorbol-12-myristate-13-acetate (PMA) results in the rearrangement of actin and tubulin filaments. PMA brings about the disorientation and diffusion of the heavily criss-crossed network of actin and microtubulin fibres characteristic of senescent cells suggesting thereby an increased sensitivity of senescent cells to phorbol esters. Since phorbol esters are known to be specific activators of protein kinase C (PKC), the PMA-induced modulation of the cytoskeleton patterns in ageing fibroblasts provides further support for the view that the effectiveness of the signalling mechanisms is retained during cellular ageing.

Altered cellular responsiveness during ageing.

The capacity of cells and organisms to respond to external stimuli and to maintain stability in order to survive decreases progressively during ageing. The mitogenic and stimulatory effects of growth factors, hormones and other agents are reduced significantly during cellular ageing. The sensitivity of ageing cells to toxic agents including antibiotics, phorbol esters, radiations and heat shock increases. This failure of homeostasis during cellular ageing does not appear to be due to any quantitative and qualitative defects in the receptor systems. Instead, metabolic defects in the pathways of macromolecular synthesis may be the basis of altered cellular responsiveness during ageing.

Homoeostatic imbalance during cellular ageing: altered responsiveness.

The inability of normal cells to maintain themselves for ever is a reflection of homoeostatic imbalance and a progressive failure of maintenance. Ageing cells respond less to growth stimulants whereas they show increased sensitivity to toxic agents including antibiotics, phorbol esters, radiation and other physical stresses. No major quantitative and qualitative defects in the receptor systems have been detected that could explain the reasons for altered responsiveness during ageing. Random metabolic defects in the processes involved in maintaining homoeostasis may be critical for causing homoeostatic imbalance, cellular ageing and death.

Reduced levels of ADP-ribosylatable elongation factor-2 in aged and SV40-transformed human cell cultures.

The elongation step is involved in the regulation of protein synthesis during the cell cycle, environmental stress, ageing and transformation. Using a diphtheria toxin-mediated assay for measuring the levels of ADP-ribosylatable elongation factor EF-2, we have observed an irreversible decrease of up to 64% in the amount of ADP-ribosylatable EF-2 in normal diploid human fibroblasts MRC-5 undergoing ageing in vitro. However, a similar decrease in low serum-associated G0/G1-arrested cells is reversible both in MRC-5 cells and in their SV40-transformed counterparts. Reduced levels of ADP-ribosylatable EF-2 could account for the slowing-down of protein synthesis during cell cycle arrest and during cellular ageing in culture.