Radiation-induced senescence-like phenotype in proliferating and plateau-phase vascular endothelial cells

The effects of ionizing radiation (IR) on tumor angiogenesis still remain largely unknown. In this study, we found that IR (8 Gy) induces a high-frequency (80-90%) senescence-like phenotype in vascular endothelial cells (ECs) undergoing exponential growth. This finding allowed us to characterize the IR-induced senescence-like (IRSL) phenotype by examining the gene expression profiles and in vitro angiogenic activities of these ECs. The expression levels of genes associated with cell cycle progression and DNA replication were remarkably reduced in the IRSL ECs. Additionally, the in vitro invasion and migration activities of these cells through Matrigel were significantly suppressed. We also found that confluent ECs exhibited a high-frequency IRSL phenotype when they were replated immediately after irradiation, whereas incubation in plateau-phase conditions reduced the induction of this phenotype and enhanced colony formation. The kinetics of DNA double-strand break repair, which showed a faster time course in confluent ECs than in growing ECs, may contribute to the protective mechanism associated with the IRSL phenotype. These results imply that the IRSL phenotype may be important for determining the angiogenic activity of ECs following irradiation. The present study should contribute to the understanding of the effects of IR on tumor angiogenesis.     

The nuclear matrix of slowly and rapidly proliferating liver cells.

The nuclear matrix of slowly proliferating rat liver is compared with rapidly proliferating regenerating liver and Zajdela ascites hepatoma cells. While no differences are detected in overall ultrastructure, composition or polypeptide profiles of normal liver versus regenerating liver matrices, significant alterations are observed in the polypeptides of Zajdela hepatoma nuclear matrices.     

A slowly proliferating subpopulation in human umbilical cord mesenchymal stem cells in culture

In this report, a slow-growing subpopulation of human umbilical cord mesenchymal stromal cells (MSCs) was identified. These cells were around 5 μm in diameter and their relative gravity was between 1.031 and 1.043 g/ml. In sharp contrast to the parent MSCs, they expressed highly CD271 and poorly the receptor for platelet-derived growth factor. Quantitative PCR with the identification of the products by DNA sequencing proved that these cells expressed Nanog at a higher level than cells from the other subpopulation (approximately 30-fold), which was further confirmed by western blotting. Furthermore, they did not grow at clonal density and depletion of these cells from the population had little effect on the colony formation of the parent MSCs. The results here indicate that a subpopulation of cells with special biological features exist in human cord MSCs in culture.     

Expression, synthesis, and phosphorylation of HSP28 family during development and decay of thermotolerance in CHO plateau-phase cells.

We investigated a correlation between development of thermotolerance and expression, synthesis, or phosphorylation of HSP28 family in CHO plateau phase cells. After heating at 45.5 degrees C for 10 min, thermotolerance developed rapidly and reached its maximum 12-18 hr after heat shock. This acquired thermal resistance was maintained for 72 hr and then gradually decayed. In parallel, the levels of three 28 kDa heat shock proteins, HSP28a along with its two phosphorylated isoforms (HSP28b,c), increased and reached their maximum 24-48 hr after heat shock. The levels of these polypeptides, except HSP28c, remained elevated for 72 hr and then decreased. The level of HSP28 mRNA increased rapidly and reached its maximum 12 hr after heat shock. However, unlike thermotolerance and the levels of HSP28 family proteins, the level of HSP28 mRNA decreased rapidly within 72 hr. These results demonstrate a correlation between the amount of intracellular HSP28 family proteins and development and decay of thermotolerance.     

Persistence of chromosomal lesions induced in actively proliferating bone marrow cells of the mouse.

The persistence of chromosomal lesions induced in vivo by mitomycin C (MMC) was evaluated by cytogenetic analysis of mouse bone marrow cells. Chromosome aberration (CA) and micronucleus (MN) frequencies were estimated at different times after treatment, up to 42 days. The frequency of CA per cell decreased in the first 3 days after treatment, but a secondary peak appeared on the 4th day, followed by a stabilization around 0.03 CA per cell (significantly different from the control value), which persisted up to 17 days. At the next time intervals tested (28 and 42 days), the CA frequency returned to the control level. In disagreement with these data obtained directly on metaphases, the MN frequency, as evaluated in polychromatic erythrocytes, decreased quickly after treatment, reaching the control value on the 5th day. We attempted to enhance the sensitivity of the MN test by using CREST antibodies and indirect immunofluorescence. However, higher proportions of CREST- MN in treated than in control animals were observed only at short time intervals, confirming the results obtained with the conventional MN assay.     

Persistence to anti-cancer treatments in the stationary to proliferating transition

The heterogeneous responses of clonal cancer cells to treatment is understood to be caused by several factors, including stochasticity, cell-cycle dynamics, and different micro-environments. In a tumor, cancer cells may encounter fluctuating conditions and transit from a stationary culture to a proliferating state, for example this may occur following treatment. Here, we undertake a quantitative evaluation of the response of single cancerous lymphoblasts (L1210 cells) to various treatments administered during this transition. Additionally, we developed an experimental system, a “Mammalian Mother Machine,” that tracks the fate of thousands of mammalian cells over several generations under transient exposure to chemotherapeutic drugs. Using our developed system, we were able to follow the same cell under repeated treatments and continuously track many generations. We found that the dynamics of the transition between stationary and proliferative states are highly variable and affect the response to drug treatment. Using cell-cycle markers, we were able to isolate a subpopulation of persister cells with distinctly higher than average survival probability. The higher survival rate encountered with cell-cycle phase specific drugs was associated with a significantly longer time-till-division, and was reduced by a non cell-cycle specific drug. Our results suggest that the variability of transition times from the stationary to the proliferating state may be an obstacle hampering the effectiveness of drugs and should be taken into account when designing treatment regimens.     

Survival of unprotected,mammalian plateau-phase cells following freezing in liquid nitrogen

Unprotected, mammalian cells in plateau phase are at least a factor of four times more sensitive to freeze-thaw damage than exponential-phase cells. The former suffer about 15–20% more sublethal damage after one freeze-thaw cycle than the latter and repair this damage more slowly. Exposure of plateau-phase cells to freeze-thaw damage lengthens the time required to traverse the cell cycle in the exposed generation. These cells may more closely represent the state in tissues than exponential-phase populations.     

Histone carbonylation occurs in proliferating cells

Chromatin is a dynamic structure formed mainly by DNA and histones, and chemical modifications on these elements regulate its compaction. Histone posttranslational modifications (PTMs) have a direct impact on chromatin conformation, controlling important cellular events such as cell proliferation and differentiation. Redox-related posttranslational modifications may have important effects on chromatin structure and function, offering a new intriguing area of research termed "redox epigenetics." Little is known about histone carbonylation, a PTM that may be related to modifications in the cellular redox environment. The aim of our study was to determine the carbonylation of the various histones during cell proliferation, a moment in cell life during which important redox changes take place. Here, we describe changes in histone carbonylation during cell proliferation in NIH3T3 fibroblasts. In addition, we have studied the variations of poly(ADP-ribosyl)ation and phospho-H2AX at the same time, because both modifications are related to DNA damage responses. High levels of carbonylation on specific histones (H1, H1(0), and H3.1 dimers) were found when cells were in an active phase of DNA synthesis. The modification decreased when nuclear proteasome activity was activated. However, these results did not correlate completely with poly(ADP-ribosyl)ation and phospho-H2AX levels. Therefore, histone carbonylation may represent a specific event during cell proliferation. We describe a new methodology named oxy-2D-TAU Western blot that allowed us to separate and analyze the carbonylation patterns of the histone variants. In addition we offer a new role for histone carbonylation and its implication in redox epigenetics. Our results suggest that histone carbonylation is involved in histone detoxification during DNA synthesis.     

Immunohistochemical detection of proliferating cells in vivo

Incorporation of the thymidine analogue 5-bromo-2'-deoxyuridine (BrUdR) into newly synthesized DNA provides the basis of a simple technique for identifying proliferating cells. BrUdR was administered to C57BL/6 mice by continuous infusion for 1-7 days, or by intraperitoneal injection for shorter intervals. Various tissue types, including gut, kidney, and liver, were excised, fixed in neutral buffered formalin, and paraffin-embedded for sectioning. De-paraffinized 4-micron tissue sections and bone marrow samples were incubated with an anti-BrUdR antibody and cells that had traversed S-phase during the BrUdR exposure period were identified immunohistochemically. Proliferation and migration of intestinal epithelial cells were identified by antibody staining after continuous in vivo exposure to BrUdR for 1-4 days, and BrUdR incorporation into proliferating marrow cells was detected within 30 min. Tissues such as normal liver, known to have low levels of proliferation, remained unstained after 3 days' exposure to BrUdR. After we established that normal proliferating cells could be identified using this technique, BrUdR was administered to mice bearing B16 melanomas. Again, proliferating tumor cells were clearly identified in histological sections. The nuclei from these paraffin-embedded tumors were also collected for flow cytometric analysis after de-waxing, rehydration, and pepsin treatment. This combination of techniques made possible the comparison in adjacent tissue sections of labeling index, obtained from stained sections, with percentage S-phase, measured using DNA flow cytometry. The % S-phase was consistently higher than the labeling index obtained with immunocytochemistry, and two-parameter DNA vs BrUdR flow cytometry showed that this difference could be accounted for by a population of unlabeled cells with an S-phase DNA content.     

Effect of thermotolerance on thermal radiosensitization in hepatoma cells

The interaction between hyperthermia and X irradiation was determined in cultured Reuber H35 hepatoma cells with different states of thermosensitivity. Incubation at 41 degrees C followed by 4-Gy X rays resulted after 2 hr in a stabilization of cell survival for heat or plus X rays, with a maximum synergism factor of 1.6. Thermotolerance did not develop during incubation at 41.7 or 42.5 degrees C. When heat treatment of cells was followed by irradiation, the synergism factor for thermal radiosensitization increased with both the amount of thermal cell killing and the amount of X-ray cell killing; the influence of thermal exposure on the synergism factor was greater than that of the X-ray dose. Cells were made thermotolerant either by incubation at 42.5 degrees C for 30 or 60 min followed by an interval at 37 degrees C, or by continuous incubation at 41 degrees C. In both cases thermotolerance was measured by incubation at 42.5 degrees C. No difference was observed between the maximum thermotolerance achieved with both methods. When cells were irradiated in addition to the second heat treatment, thermal radiosensitization was strongly reduced concomitant with the decreased sensitivity to killing by heat.     

Enhanced glycosyltransferase activity during thermotolerance development in mammalian cells

The cellular heat shock response leads to the enhanced synthesis of a family of heat shock proteins and the development of thermotolerance. In CHO cells, however, heat shock also leads to enhanced synthesis of a 50 kD glycoprotein and elevated activity of N-acetylgalactosaminyltransferase (GalNAcT). In this study we showed increased GalNAcT activity during thermotolerance expression in all of five mammalian cell lines included in the study. However, there was no simple correlation between cellular heat sensitivity of unheated control cells and basal levels of GalNAcT activity, measured toward the same exogenous acceptor apomucin. Although GalNAcT was elevated in thermotolerant cells, GalNAcT activity itself did not exhibit thermotolerance in terms of reduced sensitivity to heat inactivation. The increase in GalNAcT activity after heating was similar in exponentially growing and plateau-phase cultures and was inhibited neither by cycloheximide nor actinomycin D. However, the inhibitors by themselves also increased GalNAcT activity in unheated control cells. Chemical inducers of thermotolerance (arsenite and diamide) increased GalNAcT activity, but the increase was modest when compared to that following hyperthermia. In addition to GalNAcT, two other glycosyltransferases with specificity for O-glycans, alpha 1,2-fucosyltransferase and alpha 2,6-sialyltransferase, also showed increased activity after hyperthermia and during thermotolerance development. Together with previously published data, these results support the hypothesis that heat-induced activation of O-glycan-specific glycosyltransferases plays a physiological role in the cellular heat shock response and in thermotolerance development.     

Intracellular localization of hyaluronan in proliferating cells.

Hyaluronan is a high molecular weight glycosaminoglycan found in the extracellular matrix of many tissues, where it is believed to promote cell migration and proliferation. It was recently shown that hyaluronan-dependent pericellular matrix formation is a rapid process that occurs as cells detach during mitosis. Growing evidence for intracellular hyaluronan in tissues in vivo, together with evidence of intracellular hyaluronan binding molecules, prompted us to examine hyaluronan distribution and uptake as well as hyaluronan binding sites in cells and their relationship to cell proliferation in vitro, using a biotinylated hyaluronan binding protein and fluorescein-labeled hyaluronan. In permeabilized smooth muscle cells and fibroblasts, hyaluronan staining was seen in the cytoplasm in a diffuse, network-like pattern and in vesicles. Nuclear hyaluronan staining was observed and confirmed by confocal microscopy and was often associated with nucleoli and nuclear clefts. After serum stimulation of 3T3 cells, there was a dramatic increase in cytoplasmic hyaluronan staining, especially during late prophase/early prometaphase of mitosis. In contrast, unstimulated cells were negative. There was a pronounced alteration in the amount and distribution of hyaluronan binding sites, from a mostly nucleolar distribution in unstimulated cells to one throughout the cytoplasm and nucleus after stimulation. Exogenous fluorescein-labeled hyaluronan was taken up avidly into vesicles in growing cells but was localized distinctly compared to endogenous hyaluronan, suggesting that hyaluronan in cells may be derived from an intracellular source. These data indicate that intracellular hyaluronan may be involved in nucleolar function, chromosomal rearrangement, or other events in proliferating cells. (J Histochem Cytochem 47:1331-1341, 1999)