Apoptosis and proliferation of growth plate chondrocytes in rabbits

The growth plates of the femoral head of Japanese white rabbits aged 5, 10, 15 and 20 weeks were stained for apoptotic and proliferating chondrocytes using the TUNEL and PCNA antibody staining techniques. Both TUNEL- and PCNA-positive chondrocytes were detected in all of the specimens. The positive ratios of both stainings were calculated for the whole plate and for the resting, proliferating and hypertrophic zones. The highest ratios in both stainings occurred in the hypertrophic zone in all age groups. With growth, the TUNEL-positive ratio increased whereas the proliferating ratio decreased. We suggest that the increase in chondrocytic death by apoptosis and the decrease in cell proliferation potential led to closure of the growth plate.     

Effect of osteogenic protein-1 on the development and mineralization of primary cultures of avian growth plate chondrocytes: modulation by retinoic acid

Osteogenic protein-1 (OP-1), a member of the TGF-beta family of proteins, induces endochondral bone formation. Here we studied the effect of OP-1 on the development of primary cultures of avian growth plate (GP) chondrocytes in either serum-free or serum-containing medium, in the absence or presence of retinoic acid (RA). OP-1 was added on day 7 of culture and continued for 7 days, or until the cultures were harvested, typically on day 21. Alone, OP-1 caused approximately 2-fold increase in proteoglycan synthesis into both the medium and the cell:matrix layer. Additionally, OP-1 caused a dosage-dependent increase in alkaline phosphatase (ALP) activity, and an increase in protein, when given from days 7-14 and examined on day 14. This stimulation was greater in cells grown in serum-free than in serum-containing media (3-5-fold vs. 2-3-fold increase in ALP; approximately 40% vs. approximately 20% increase in protein). Such stimulation of ALP activity and proteoglycan (PG) synthesis in cultured GP cells indicates that OP-1 elicits differentiation of chondrocytes. OP-1 minimally affected cell division (DNA content); however, a slight increase was seen when examined early in the culture. Alone, OP-1 increased mineral (Ca and Pi) content of the cultures by approximately 2-fold in both types of media. As early as day 14, clusters of mineral encircled many of the OP-1 treated cells. Thus, as in vivo, OP-1 strongly promoted mineral formation by the cultured GP chondrocytes. When present together, OP-1 and RA generally blocked the action of the other. Separately OP-1 and RA each stimulated protein synthesis, ALP activity, and Ca2+ deposition; together they were inhibitory to each. Also, RA blocked the stimulation of PG synthesis induced by OP-1; whereas OP-1 decreased cell division engendered by RA. Thus, this GP chondrocyte culture system is a good model for studying factors that influence differentiation and mineral deposition during bone growth in vivo.     

Retinoic acid stimulates matrix calcification and initiates type I collagen synthesis in primary cultures of avian weight-bearing growth plate chondrocytes

The effect of retinoic acid (RA) on primary cultures of growth plate chondrocytes obtained from weight-bearing joints was examined, Chondrocytes were isolated from the tibial epiphysis of 6- to 8-week-old broiler-strain chickens and cultured in either serum-containing or serum-free media. RA was administered at low levels either transiently or continuously after the cells had become established in culture. Effects of RA on cellular protein levels, alkaline phosphatase (AP) activity, synthesis of proteoglycan (PG), matrix calcification, cellular morphology, synthesis of tissue-specific types of collagen, and level of matrix metalloproteinase (MMP) activity were explored. RA treatment generally increased AP activity and stimulated mineral deposition, especially if present continuously. RA also caused a shift in cell morphology from spherical/polygonal to spindle-like. This occurred in conjunction with a change in the type of collagen synthesized: type X and II collagens were decreased, while synthesis of type I collagen was increased. There was also a marked increase in the activity of MMP. Contrasting effects of continuous RA treatment on cellular protein levels were seen: they were enhanced in serum-containing media, but decreased in serum-free HL-1 media. Levels of RA as low as 10 nM significantly inhibited PG synthesis and caused depletion in the levels of PG in the medium and cell-matrix layer. Thus, in these appendicular chondrocytes, RA suppressed chondrocytic (PG, cartilage-specific collagens) and enhanced osteoblastic phenotype (cell morphology, type I collagen, alkaline phosphatase, and mineralization).     

Monoclonal antibody enhanced stimulation of human growth hormone action in vitro using ovine costal cartilage growth plate chondrocytes

Investigations into the mechanism underlying antibody-mediated enhancement of growth hormone action have been hampered by the lack of an in vitro assay system. In this work, we have used isolated ovine costal cartilage growth plate chondrocytes to demonstrate, for the first time, that monoclonal antibody EB1 can enhance the proliferative actions of human growth hormone on this cell type. Chondrocytes were cultured for 14 days prior to exposure to GH+/-monoclonal antibody EB1 for a 4-day treatment period. Human growth hormone alone promoted a significant dose-dependent increase in chondrocyte proliferation; maximal stimulation was achieved at about 3.3-10 ng/ml growth hormone, and at higher doses of growth hormone, the response declined. Monoclonal antibody EB1 was shown to enhance the proliferative activity of 10 ng/ml human growth hormone in a significant dose dependent fashion. In conclusion, our results demonstrate that an antibody capable of enhancing GH activity in vivo also has the capacity to potentiate GH activity in vitro. This system may provide an important tool for investigations into the mechanism of GH action, and how this is modified by GH enhancing MAbs.     

Identification and cloning of a novel phosphatase expressed at high levels in differentiating growth plate chondrocytes

Growth plate chondrocytes progress through a proliferative phase before acquiring a terminally-differentiated phenotype. In this study we used Percoll density gradients to separate chick growth plate chondrocytes into populations of different maturational phenotype. By applying agarose gel differential display to these populations we cloned a cDNA encoding a novel 268 amino acid protein (3X11A). 3X11A contains two peptide motifs that are conserved in a recently identified superfamily of phosphotransferases. It is likely that 3X11A is a phosphatase, but its substrate specificity remains uncertain. 3X11A expression is upregulated 5-fold during chondrocyte terminal differentiation and its expression is approximately 100-fold higher in hypertrophic chondrocytes than in non-chondrogenic tissues. This suggests that 3X11A participates in a biochemical pathway that is particularly active in differentiating chondrocytes.     

In situ levels of intracellular Ca2+ and pH in avian growth plate cartilage

Interactions with the extracellular matrix, accumulation of Ca2+, formation of matrix vesicles, and regulation of tissue pH by growth plate chondrocytes all appear to be vital to endochondral calcification. Thus, the activities of Ca2+ and H+ ions in these cells, while still embedded in their organic matrix, are of great interest. Using laser confocal imaging and sensitive Ca2+ (Indo 1) and pH (BCECF) probes, cellular Ca2+ and pH were analyzed in thin sections of freshly isolated cartilage. Mean values of cytosolic Ca2+ in cells from the various zones of the growth plate were quite similar, but levels in individual cells and subcellular compartments varied significantly. Ca2+ was elevated intensely near the periphery of cells in the zones of maturation and hypertrophy, and many Ca2+ rich particles were seen in the matrix near these cells. Levels of Ca2+ within the cells varied with time. In the proliferative region, cyclical increases and decreases in Ca2+ were seen, but there was little shedding of Ca2+ rich particles. However, after repeated Ca2+ cycling, in the zones of maturation and hypertrophy, Ca2+ rich particles were shed from the cell surface, forming what appeared to be matrix vesicles. Intracellular pH levels also varied significantly within the chondrocytes and between the cells and zones. Numerous focal elevations in pH (> 8.0) were seen in central regions of the maturing and early hypertrophic cells, with lower pH (6.5-7.2) near the cell periphery of the late hypertrophic and calcifying cells. This pattern of cytoplasmic alkalinization and subsequent acidification appears to contribute to loading of Ca2+ and Pi into matrix vesicles during their formation by the chondrocytes.     

Death activation does not stop tumor growth: quantitative evidence for concomitant activation of apoptosis in tumor growth in vitro

A protein-independent fibrosarcoma, Gc-4 PF, grows exponentially in a protein-free medium. The doubling time (approximately 26 h) was similar to that of the serum-dependent parental clone, Gc-4 SD cultivated in the presence of fetal calf serum (FCS). We demonstrated here that the protein-free cultivation of Gc-4 PF cells concomitantly activates apoptotic phenotypes (one third of total cell population), including typical morphology, high uptake of Hoechst 33342 dye, and cleavage of DNA to large fragments, as observed in protein-deprived Gc-4 SD cell previously. Gc-4 SD cells arrested in the G0/G1-phase in response to the protein-free condition. In contrast, Gc-4 PF cells did not reach G0/G1 arrest in the protein-free condition; instead the durations of both G0/G1 and G2-phases were markedly reduced. The estimation of one cell cycle duration revealed that the cell division cycle was accelerated to 1.7 (27 h/15.4 h)-fold. Then the growth kinetics was able to be verified quantitatively by both the cell division rate and apoptotic cell loss. Protein-free cultivation resulted in slight down-regulation of c-myc protein in both cell types, while the down-regulation of p34cdc2, shown clearly in Gc-4 SD cells, was avoided in Gc-4 PF cells. Interestingly, while the expression of p53 was not affected in Gc-4 SD cells in response to the protein-free condition, the suppressor gene product expression was suppressed markedly in Gc-4 PF cells. These results suggest that Gc-4 PF cells may have acquired an ability to accelerate cell division by shortening the cell cycle duration to maintain a proper growth rate in response to intrinsic apoptosis activation with, at least in part, a suppression of p53 expression as well as an escape of down-regulation of p34cdc2.     

Subjective interpretation, laboratory error and the value of forensic DNA evidence: three case studies

This article discusses two factors that may profoundly affect the value of DNA evidence for proving that two samples have a common source: uncertainty about the interpretation of test results and the possibility of laboratory error. Three case studies are presented to illustrate the importance of the analyst's subjective judgments in interpreting some RFLP-based forensic DNA tests. In each case, the likelihood ratio describing the value of DNA evidence is shown to be dramatically reduced by uncertainty about the scoring of bands and the possibility of laboratory error. The article concludes that statistical estimates of the frequency of matching genotypes can be a misleading index of the value of DNA evidence, and that more adequate indices are needed. It also argues that forensic laboratories should comply with the National Research Council's recommendation that forensic test results be scored in a blind or objective manner.     

Evidence of significant apoptosis in poorly differentiated ductal carcinoma in situ of the breast

Following breast-conserving surgery for ductal carcinoma in situ (DCIS), the presence of comedo necrosis reportedly predicts for higher rates of post-operative recurrence. To examine the role of programmed cell death (apoptosis) in the aetiology of the cell death described as comedo necrosis, we studied 58 DCIS samples, using light microscopy, for morphological evidence of apoptotic cell death. The percentage of apoptotic cells (apoptotic index, AI) was compared between DCIS with and without evidence of 'comedo necrosis' and related to the immunohistochemical expression of the anti-apoptosis gene bcl-2, mitotic index (MI), the cellular proliferation antigen Ki67, nuclear grade and oestrogen receptor (ER) status. AI was significantly higher in DCIS samples displaying high-grade comedo necrosis than in low-grade non-comedo samples: median AI = 1.60% (range 0.84-2.89%) and 0.45% (0.1-1.31%) respectively (P < 0.001). Increasing nuclear grade correlated positively with AI (P < 0.001) and negatively with bcl-2 expression (P = 0.003). Bcl-2 correlated negatively with AI (P = 0.019) and strongly with ER immunoreactivity (P < 0.001). Cellular proliferation markers (MI and Ki67 immunostaining) correlated strongly with AI and were higher in comedo lesions and tumours of high nuclear grade (P < 0.001 in all cases). Thus, apoptosis contributes significantly to the cell death described in ER-negative, high-grade DCIS in which a high proliferative rate is associated with a high apoptotic rate. It is likely that dysregulation of proliferation/apoptosis control mechanisms accounts for the more malignant features typical of ER negative comedo DCIS.     

Measurement of cell proliferation by labeling of DNA with stable isotope-labeled glucose: studies in vitro, in animals, and in humans

A method for measuring DNA synthesis and, thus, cell proliferation, in vivo is presented. The technique consists of administering [6,6-2H2]Glc or [U-13C]Glc, isolating genomic DNA, hydrolyzing enzymatically to free deoxyribonucleosides, and derivatizing for GC-MS analysis of dA or dG isotopic enrichments, or both. Comparison of dA or dG to extracellular Glc enrichment (with a correction for intracellular dilution) reveals the fraction of newly synthesized DNA, by application of the precursor-product relationship. Thus, the technique differs from the widely used [3H]thymidine or BrdUrd techniques in that the de novo nucleotide synthesis pathway, rather than the nucleoside salvage pathway, is used to label DNA; the deoxyribose rather than the base moiety is labeled; purine rather than pyrimidine deoxyribonucleosides are analyzed; and stable isotopes rather than radioisotopes are used. The method is applied here in vitro to the growth of HepG2 and H9 cells in culture; in animals to proliferation of intestinal epithelium, thymus, and liver; and in humans to granulocyte turnover in blood. In all instances, measured cell proliferation kinetics were consistent with expected or independently measured kinetics. The method has several advantages over previously available techniques for measuring cell turnover, involves no radioactivity or potentially toxic metabolites, and is suitable for use in humans. The availability of a reliable and safe method for measuring cell proliferation in humans opens up a number of fundamental questions to direct experimental testing, including basic problems related to cancer, AIDS, and other pathologic states.     

Determinants of spatial polarity in the growth plate

Growth of long bones occurs at the growth plate, a layer of cartilage that separates the epiphysis from the metaphysis. Growth plate exhibits spatial polarity. Proliferative chondrocytes undergo terminal differentiation when they approach the metaphyseal, but not the epiphyseal, border of the growth plate. The adjacent bone also exhibits spatial polarity. Metaphyseal, but not epiphyseal, blood vessels and bone cells invade the adjacent growth plate, remodeling it into bone. As a result, the metaphysis, but not the epiphysis, elongates over time. To determine whether cartilage polarity determines bone polarity and/or whether bone polarity determines cartilage polarity, rabbit distal ulnar growth plates were excised, inverted, and reimplanted in their original beds. Thus, cartilage polarity was inverted relative to bone polarity. Histological examination showed that the inverted cartilage polarity was maintained over time. In contrast, the polarity of the adjacent bone reversed after surgery, to match that of the cartilage. Blood vessel and bone cell invasion ceased in the metaphysis and arose in the epiphysis. Longitudinal bone growth (measured with weekly radiographs) occurred at the epiphyseal, not at the metaphyseal, surface of the growth plate. We conclude that the polarity of growth plate cartilage is determined by intrinsic factors. The cartilage polarity then determines the polarity of the adjacent bone and, consequently, the functional polarity of longitudinal bone growth.     

Effect of methylglyoxal on human leukaemia 60 cell growth: modification of DNA G1 growth arrest and induction of apoptosis

Results of laboratory experiments demonstrating that ants are capable of assessing the number of objects within the limits of several tens and transmit this information to other individuals are described. It has been demonstrated that ants may use these capacities for transmitting information regarding the coordinates of an object.     

In situ detection of apoptosis in regressing corpus luteum of pregnant sow: evidence of an early presence of DNA fragmentation

Luteolysis has been shown to be correlated with apoptosis in rats, sheep, and cows. In pigs, apoptosis has already been demonstrated as regards atretic follicles. The present study has been conducted to evaluate whether apoptosis occurs during corpora lutea regression in the pregnant pig and to investigate the temporal relationship between apoptosis and functional luteolysis. The apoptotic process has been studied through the research of oligonucleosome fragmentation by means of classical electrophoresis methods and by in situ detection on histological luteal sections. The latter method allows the identification of apoptosis and the localization of apoptotic cells. Pregnant sows were cloprostenol (PGF2 alpha analog) treated and ovariectomized 0, 6, 12, 24, 48, and 72 hr after treatment. Corpora lutea were utilized for progesterone and DNA extraction and in situ evaluation of apoptosis. Clear evidence of apoptosis was seen earlier with the in situ technique (6 hr for stromal tissue, 12 hr for luteal cells) than with the classical method (24 hr). Apoptosis was, however, apparent after plasma and tissue progesterone had reached basal levels. In conclusion, these results are consistent with the hypothesis that apoptosis occurs during luteolysis in pigs. Moreover, the data obtained with the in situ technique made it possible to identify signs of structural regression in stromal tissue first than in parenchymal cells. A two-stage activation of apoptosis has been discussed to explain structural changes that occur during luteolysis after cloprostenol treatment in swine corpora lutea.     

Basic fibroblast growth factor and insulinlike growth factor I support the growth of human septal chondrocytes in a serum-free environment

OBJECTIVE: To determine if insulinlike growth factor I (IGF-I) and basic fibroblast growth factor (bFGF), individually or in combination, support the growth and viability of human septal chondrocytes in a serum-free medium (SFM) and a serum-enhanced culture medium. DESIGN: Chondrocytes were recovered from enzymatically digested human septal cartilage and were plated for monolayer culture in a newly developed medium. The medium included Dulbecco modified Eagle medium mixed 1:1 with Ham F12 medium and a supplement of known amounts of 2 growth factors-bFGF (100 ng/mL) and IGF-I (100 ng/mL)-used in combination and separately. RESULTS: The combination of IGF-I and bFGF enhanced chondrocyte growth and maintained a high degree of viability in SFM and 10% fetal calf serum. After an initial lag, the SFM, augmented with both growth factors, produced a comparable number of viable cells (4.25+/-0.31 x 10(4)) to that of the medium with 10% fetal calf serum (4.64+/-0.35 x 10(4)) by the seventh day of the experiment. Combined with the 2 growth factors, 10% fetal calf serum provided the greatest proliferation by the end of the experiment. However, the overall mean cell counts for the IGF-I- and bFGF-enhanced SFM were not statistically different. CONCLUSIONS: The combination of IGF-I and bFGF in a serum-free and a serum-supplemented environment supports the growth and viability of human septal chondrocytes in short-term culture. In an SFM, the results obtained approximate those produced in a medium enhanced with 10% fetal calf serum.     

LCR-dependent gene expression in beta-globin YAC transgenics: detailed structural studies validate functional analysis even in the presence of fragmented YACs

Yeast artificial chromosome (YAC) transgenesis is associated with a high frequency of deletions in the integrated transgenes. To determine the impact of these rearrangements on the ability to derive structure-function relationships using YACs, transgenic mice were generated with 248 or 155 kb beta-globin locus YACs. The transgenics were examined for structural integrity of the YAC using an approach of structural analysis that unambiguously demonstrates intactness of YAC transgene copies. Globin gene expression per copy of each integrated transgene and the profiles of globin gene expression during development were determined. Diverse deletion patterns were observed in one or more integrated YACs in all the 248 and most of the 155 kb transgenic lines we analyzed. However, when the structure of the major regulatory element of the beta-globin locus, the locus control region, was preserved, the genes of the beta-globin locus functioned normally and globin transgenes of both the 248 and 155 kb beta-YACs were expressed in a position-independent, copy number-dependent manner. Furthermore, the globin genes of both beta-YACs displayed normal developmental regulation. We conclude that YACs can be used for analysis of structure-function relationships of large genes or multigene loci in spite of the tendency for rearrangements and deletions of the integrated transgenes. However, detailed structural evidence for integrity and continuity of locus sequences is required for correct interpretation of functional data.     

Use of live oocyst vaccines in the control of avian coccidiosis: experimental studies and field trials

Areas addressed in this study on the use of live oocyst vaccines to control coccidiosis include: the influence of immunocompetency of the strains and sex of the birds used; methods of delivery of vaccine; immunological variation between different strains of the same coccidial species; and the effects of combining vaccine with anticoccidial medication. The results show that vaccination with live oocysts elicited significant protection against coccidiosis, both with experimentally induced and naturally acquired coccidial infection, resulting in average bird weight gains and feed efficiency similar to that obtained with conventional anticoccidial medication.     

Experimental study on growth of epiphysial plate: free graft in rabbits

The growth potential of a free graft of an epiphysial plate was investigated in rabbits. Two epiphysial plate grafts were harvested from each iliac crest. One was grafted to the head (onto bone) and the other to the ear (onto cartilage). Both of the epiphysial plates enlarged to a maximum height of 1.4 cm and became similar to iliac crests. Enchondral ossification was observed up till approximately 28 weeks of age. We conclude that an epiphysial plate has growth potential after free heterotopic transplantation.     

Apoptosis in renal cell carcinoma: detection by in situ end-labeling of fragmented DNA and correlation with other prognostic factors

This study examined the acute effects of oral inhalation of 10-ppm hydrogen sulfide (H2S) inhalation (a concentration equal to its occupational exposure limit) on the pulmonary function in healthy men and women. Nine men and ten women consented to inhale medical air or 10 ppm H2S for 15 minutes each during cycle exercise at 50% of their maximal aerobic power. Routine pulmonary function tests were administered at rest and immediately after the two exposure conditions. The results indicated no significant changes in any of the variables derived from the flow volume loop, maximum ventilation volume, and diffusion capacity of the lung for carbon monoxide in both genders. None of the subjects experienced any signs and symptoms as a result of H2S exposure. It was concluded that oral inhalation of 10 ppm H2S at an elevated metabolic and ventilation rate does not significantly alter pulmonary function in healthy men and women.