In vitro thymidine labelling in human and porcine growth plates

An in vitro technique has been used to label dividing cells in the growth plates of human bones with tritiated thymidine. The patterns of labelling in autoradiographs of human plates are described and values given for the labelling index, the number of cells in the proliferation zones and the heights of hypertrophied cells. In two of the four subjects no labelled cells were found in the growth plates and possible causes for these failures are discussed. In vitro labelling data on five porcine growth plates are also presented for comparison with the human data. In both structure and cell kinetics the epiphyseal cartilage plate in the pig is intermediate between the human and rodent plates.     

A comparison of different methods to determine cell proliferation by flow cytometry

Abstract. The proliferation of human melanoma cells (MeWo) in vitro was studied with a number of different techniques. In particular, we compared the expression of PCNA and the Ki-67 antigen on the one hand with BrdU pulse and continuous labelling on the other. Two-dimensional flow cytometry (with DNA content as a second parameter) was employed to discriminate between cycling and non-cycling cells as well as cells in the G₁, S and G₂ phases of the cycle. Cell cultures in different stages of growth were analyzed. We found that the percentage of anti-PCNA and Ki-67 positive cells agreed very well with the BrdU pulse and continuous labelling index, respectively. Our data further support the assumption that under certain conditions PCNA is a marker of S-phase cells, whereas Ki-67 can be used to quantify the growth fraction. Possible pitfalls of the techniques are discussed.     

Growth Fraction of Myelocytes In Normal Human Granulopoiesis

Abstract. The labelling index (LI) of myelocytes (M) after flash labelling of normal human bone marrow cells with [³H]-thymidine ([³H]TdR) is always lower that the LI obtained for myeloblasts (MB) and for promyelocytes (PM). This fact can be interpreted in two ways: it may mean that the duration of the G₁ phase of the cell cycle is longer in M than in MB or PM, or it may mean that the proportion of cells in cycle, i.e., the growth fraction (GF), is lower in the M population than in MB or PM. the evolution of the LI and of the mean number of grains per cell was monitored in [³H]TdR-labelled normal bone marrow during in vitro incubation for 50 hr. the generation time, measured by the halving time of the mean number of grains per cell after flash labelling, was similar for M to that for MB and PM. During continuous labelling, the LI of MB and PM reached 1 and the LI value for M never rose to more than 50% of the values recorded for MB and PM after 30 hr. These findings give support to the second hypothesis, i.e., a lower GF in the M population. Good correlation was found between the LI of M and the proportion of mature polymorphonuclear cells in the bone marrow of normal subjects and of patients with chronic benign neutropenia or hyperleucocytosis. Variations in the M growth fraction could be a medium-term (2-3 days) regulatory factor in granulocyte production.     

Detailed Kinetic Analysis of Shay Chloroleukaemia Cell Population Propagated In Permanent Suspension Culture In Vitro

Detailed kinetic analysis of a growing cell population is difficult, even when assay conditions are nearly ideal. Therefore, it is usually essential to perform several types of experiments and analyse all the results in terms of a mathematical model, the use of which is not limited a priori by a specified application. In the present study we investigated cell population kinetics using rat chloroleukaemia cells propagated in suspension culture in vitro. the parameters were measured: doubling time of the population, fraction of labelled mitoses, changes in labelling index with time after pulse labelling, continuous labelling and stathmokinetic index. Analysis of the results was based on a computer program CECAM, which is a stochastic model capable of simulating essentially all types of kinetic experiments based on presently known assay techniques. The results showed that precise and reliable information on cell population kinetics could not be obtained from the analysis of any single type of experimental data. In particular, the technique of labelled mitoses underestimated the duration of the G₁ phase, owing to subtle label-induced changes in population behaviour. These changes could not have been detected with any certainty without rigorous quantitative comparisons with other types of experimental data. As a whole, however, results obtained by the different techniques were in agreement and the kinetic behaviour of chloroleukaemia cells in vitro could be established in detail. In certain circumstances even minute changes in the kinetic parameters of the cells can modify population behaviour drastically. to study these cases adequately the experiments must be designed with utmost care, preferably with the aid of preceding simulations. This is because demonstration of small primary changes in population kinetics may be beyond the limit of detection of any single assay method.     

In situ photolabelling of the human androgen receptor

In situ photoaffinity labelling of the human androgen receptor has been performed in the LNCaP (Lymph Node Carcinoma of the Prostate) cell line. The covalently labelled receptors were identified by SDS-PAGE. Intact LNCaP cells, incubated with [3H]-R1881 and subsequently irradiated with u.v. light and directly solubilized in SDS-buffer, revealed two photolabelled protein bands at 110 and 50 kDa. Irradiation of intact cells and subsequent isolation of nuclei followed by extraction with 0.5 M NaCl resulted in one major photolabelled protein band at 110 kDa. The labelling of this band could be completely suppressed by a 100-fold molar excess of non-radioactive R1881. Photolabelling of androgen receptors in a cytosolic preparation of LNCaP cells after anion exchange chromatography resulted in a much lower labelling efficiency compared with the in situ labelling procedure, although the androgen receptor was purified 100-fold. The steroid binding domain of the human androgen receptor has been partially mapped with chymotrypsin and S. aureus V8 protease digestion. Proteolytic digestion with chymotrypsin of purified photoaffinity-labelled 110 kDa human androgen receptor resulted in the generation of a 15 kDa peptide which still contains the covalently linked hormone. It is concluded that the in situ photoaffinity labelling technique can be applied successfully for characterization of the steroid binding domain of androgen receptors in prostate cancer cells and in other androgen target cells. Furthermore, it was demonstrated that the human androgen receptor is a monomer with a molecular mass of 110 kDa, of which the steroid binding site is confined to a 15 kDa domain.     

Influence of Growth Hormone and Thyroxine On Cell Kinetics In the Proximal Tibial Growth Plate of Snell Dwarf Mice

Abstract. In Snell dwarf mice, the influence of short-term treatment with human growth hormone (hGH) or thyroxine on the proliferative and sulphation activity of the proximal tibial growth plate was studied. By autoradiographic methods, the [³H]methylthymidine incorporation after a single injection was measured, after 2 hr incorporation time. the labelling index was calculated and the number of labelled mitoses was counted. In addition, the distribution of the labelled nuclei over the proliferating and degenerating zones was determined by continuous labelling for 25 and 73 hr.
In untreated dwarf mice after [³H]-methylthymidine administration, the number of labelled nuclei in the growth plate is low. Labelling occurs, as expected, mainly in the cells of the proliferative zones. the number of labelled nuclei in control dwarf mice was similar after 25 and 73 hr continuous labelling. This suggests that many cells are in a resting G_o or prolonged G₁ phase. Both hGH and T₄ treatment induce a significant increase of the number of labelled nuclei per growth plate and of the number of mitoses. Since hormonal treatment induces a small number of mitoses after 2 hr incorporation of the label, the minimal G₂ phase of the cell cycle is less than 2 hr. In addition, treatment with hGH and T₄ stimulates chondrocytes in the zone of proliferative and hypertrophic cells to actively incorporate [³⁵S]-sulphate.     

In vitro determination of myeloma cell resistance to melphalan using the 3H-thymidine incorporation technique.

Plasma cells derived from marrow aspirates of 21 untreated myeloma patients have been cultured in RPMI 1640 medium containing 3H-thymidine and melphalan. Thereafter plasma cell-labelling index was determined autoradiographically. In 20 patients melphalan caused a measurable decrease in the percentage of labelled plasma cells. Decreases in labelling index ranging from -4% to 3% have been considered as indicating the resistance of myeloma cells to melphalan in vitro. Decreases within this range have been found in 7 patients. In other 13 myelomas with chemosensitive marrow plasma cells, melphalan did reduce the labelling index to values ranging from 4% to 24%. Some clinical implications of the tests are discussed.     

In situ detection of the mast cell proteases chymase and tryptase in human lung tissue using light and electron microscopy

Scroll-rich, "mucosal" mast cells are the predominant human lung mast cell type. It has been proposed that these mast cells store tryptase but are mostly chymase deficient. We present a detailed immunolocalisation study of chymase and tryptase in lung specimens of eight patients. Using monoclonal antibody B7 in a conventional tissue processing method for light microscopy, chymase-positive mast cells were much fewer than tryptase-positive ones. However, they approached the number of tryptase-positive cells when optimised processing was used. Two different monoclonal antibodies, B7 and CC1, were used to visualise chymase in purified lung mast cells of two patients using ultrastructural immunogold labelling. Immunoabsorption controls demonstrated a reactivity of B7 with both tryptase and chymase, but indicated specificity of CC1 for chymase. On the ultrastructural level, all of more than 1,400 lung mast cells evaluated labelled for chymase. Reactivity was seen in cytoplasmic granules, cytoplasm and vesicles, but not elsewhere. Tryptase labelling using monoclonal antibody G3 was also present in all mast cells detected, and was retained in altered granules (=activated mast cells), where B7 labelling was sparse. The average labelling density was approximately sixfold higher than for chymase. In summary, chymase may be more abundant in human lung mast cells than hitherto thought.     

The labelling index of human and mouse tumours assessed by bromodeoxyuridine staining in vitro and in vivo and flow cytometry

Bromodeoxyuridine (BrdUrd) incorporation and flow cytometry were used to measure human tumour kinetic parameters in vitro and in vivo. The technique was validated by comparison of labelling index estimates of mouse tumours in vivo and in vitro using BrdUrd and flow cytometry with tritiated thymidine (3HdThd) autoradiography. Similar labelling indices were obtained with both in vivo and in vitro incorporation into DNA of the two different precursors. Measurements of human tumour labelling indices were similar following in vitro incubation with either BrdUrd or 3HdThd. The use of BrdUrd allowed the visualization of a population of S-phase cells that did not appear to incorporate BrdUrd or 3HdThd. The human tumour labelling indices obtained with BrdUrd incorporation were similar to previously reported values using autoradiography studies. Preliminary studies demonstrated that significant human tumour labelling could be achieved with an intravenous injection of 500 mg BrdUrd.     

Platelet-derived growth factor receptor-β-positive telocytes in skeletal muscle interstitium

Telocytes (TCs) represent a new cell type recently described in mammalian skeletal muscle interstitium as well as in other organs. These have a specific morphology and phenotype, both in situ and in vitro. Telocytes are cells with long and slender cell prolongations, in contact with other interstitial cells, nerve fibres, blood capillaries and resident stem cells in niches. Our aim was to investigate the potential contribution of TCs to micro-vascular networks by immunofluorescent labelling of specific angiogenic growth factors and receptors. We found that in human skeletal muscle TCs were constantly located around intermediate and small blood vessels and endomysial capillaries. Epi-fluorescence and laser confocal microscopy showed that TCs express c-kit, platelet-derived growth factor receptor (PDGFR)-β and VEGF, both in situ and in vitro. Telocytes were constantly located in the perivascular or pericapillary space, as confirmed by double staining of c-kit/CD31, PDGFR-β/CD31 and PDGFR-β/α-smooth muscle actin, respectively. Electron microscopy (EM) differentiated between pericytes and other cell types. Laminin labelling showed that TCs are not enclosed or surrounded by a basal lamina in contrast to mural cells. In conclusion, a) PDGFR-β could be used as a marker for TCs and b) TCs are presumably a transitional population in the complex process of mural cell recruitment during angiogenesis and vascular remodelling.     

Mitotic and Labelling Activity In Normal Human Epidermis In Vivo

Labelling and mitotic indices were studied in the epidermis of twenty-eight young men. A mean labelling index of 5.5% was found from the whole study and a mean mitotic index of 0.06%. Mitotic index particularly was extremely variable; indices between 0.002 and 0.438% were found in individual biopsies. In the first two of three experiments in which mitotic index at 09.00 hours was compared with that at 15.00 hours, significant differences were found (15.00 hours > 09.00 hours by a factor of 2.6, P < 0.001). However, in the third such experiment no such difference was found, suggesting that the timing and occurrence of diurnal rhythms of mitotic activity may not be consistent in normal human epidermis. In the one experiment in which it was investigated, a significantly higher mitotic index was found at 21.00 hours compared to 09.00 and 15.00 hours. Labelling index did not vary significantly at 09.00, 15.00 or 21.00 hours. However, labelling index did show a significant pattern of change over a 12-month period in two groups of subjects; peaks of labelling were seen in July and troughs in January. Very high ratios of labelled: mitotic cells were found, the median ratio for the whole study being ninety-eight labelled: one mitotic cell. This finding supports the possibility that not all labelled cells subsequently go on to divide in normal human epidermis.     

In vitro cytotoxicity assays. Potential alternatives to the Draize ocular allergy test

A short-term cytotoxicity assay carried out in multiwell test plates and a supplementary colony forming assay are both useful for screening and range finding of toxic concentrations of test agents. The highest tolerated dose (HTD), a concentration at which only minimal morphological changes were observed, was designated as endpoint in the assay. Epithelial rabbit cornea cells, murine fibroblasts, Chinese hamster lung cells, human hepatoma cells and mouse macrophage cultures were used as targets. Several of the alcohols tested at HTD in the colony forming assay were found to inhibit colony formation. An ID50 of colony formation was used as a quantitative corroborating test. The ranking of 34 toxicants was found to be virtually the same with all cell types examined. This easily reproducible, rapid in vitro test is cost-effective and can be used for preliminary large scale screening of potential toxicants.Abbreviations HTD highest tolerated dose - EGF epidermal growth factor - FITC fluorescein conjugated anti-guinea pig IgG - DMEM Dulbecco's modified Eagle's medium - FBS fetal bovine serum Supported in part by Revlon, Inc.     

In vitro differentiation of human mesenchymal stem cells to epithelial lineage

Our study examined whether human bone marrow-derived MSCs are able to differentiate, in vitro, into functional epithelial-like cells. MSCs were isolated from the sternum of 8 patients with different hematological disorders. The surface phenotype of these cells was characterized.To induce epithelial differentiation, MSCs were cultured using Epidermal Growth Factor, Keratinocyte Growth Factor, Hepatocyte Growth Factor and Insulin-like growth Factor-II. Differentiated cells were further characterized both morphologically and functionally by their capacity to express markers with specificity for epithelial lineage. The expression of cytokeratin 19 was assessed by immunocytochemistry, and cytokeratin 18 was evaluated by quantitative RT-PCR (Taq-man). The data demonstrate that human MSCs isolated from human bone marrow can differentiate into epithelial-like cells and may thus serve as a cell source for tissue engineering and cell therapy of epithelial tissue.     

Parabolic growth patterns in 96-well plate cell growth experiments

Summary In preparing for the routine use of the ubiquitous in vitro cell growth inhibition assay for the study of anticancer agents, we characterized the statistical properties of the assay and found some surprising results. Parabolic well-to-well cell growth patterns were discovered, which could profoundly affect the results of routine growth inhibition studies of anticancer and other agents. Four human ovarian cell lines, A2780/WT, A2780/DX5, A2780/DX5B, and A121, and one human ileocecal adenocarcinoma cell line, HCT-8, were seeded into plastic 96-well plates with a 12-channel pipette, without drugs, and grown from 1–5 d. The wells were washed with a plate washer, cells stained with sulforhodamine B (SRB), and dye absorbance measured with a plate reader. Variance models were fit to the data from replicates to determine the nature of the heteroscedastic error structure. Exponential growth models were fit to data to estimate doubling times for each cell line. Polynomial models were fit to data from 10-plate stacks of 96-well plates to explore nonuniformity of cell growth in wells in different regions of the stacks. Each separate step in the assay was examined for precision, patterns, and underlying causes of variation. Differential evaporation of water from wells is likely a major, but not exclusive, contributor to the systematic well-to-well cell growth patterns. Because the fundamental underlying causes of the parabolic growth patterns were not conclusively found, a randomization step for the growth assay was developed.     

In vitro bactericidal action of a complex of natural cytokins on Streptococcus pyogenes

The data on the antimicrobial in vitro action of a complex of natural cytokins (CNC)--the substance and the preparation "Superlymph"--on virulent museum and clinical macrolid-resistant group A beta-hemolytic streptococci, are presented. CNC suppressed the growth of museum test culture of streptococci in the concentration dependent manner. Moreover, the preparation "Superlymph" proved to be an effective antistreptococcal remedy in its action on erythromycin-resistant group A streptococcal (GAS) strains. The substance of the preparation induced the lysis of GAS cells, grown on lawn plates and in the system of spectrophotometric analysis. The effect thus established could be caused by cationic antimicrobial peptides (protegrins), contained in "Superlymph".     

The effect of dibromodulcitol on resting and dividing lymphoid cells

The effect of dibromodulcitol (DBD) on the incorporation of labelled precursors into DNA and RNA fractions of PHA-stimulated human lymphocytes and of P388F lymphoma cells at various stages of their growth was studied. Both cell systems showed sensitivity to the drug within the concentration range of 1–10 μg/ml.When DBD was added before phytohaemagglutinin (PHA), human lymphocytes showed a DNA labelling that was more affected than RNA. In contrast, by adding DBD after PHA, RNA labelling was much more inhibited than DNA. In the latter case, the decrease in DNA labelling occurred only 24 h after drug treatment whereas RNA labelling was decreased 1 h after treatment. Levels of DBD which normally produced 30% inhibition in plating efficiency of P388F lymphoma cells affected uridine-5-T incorporation to a different extent at different stages of growth of the culture. Enhanced RNA labelling occurred in early exponential stage while at later stages of growth, RNA synthesis was depressed.     

In situ ultrastructural localization of sugar-binding sites in lizard granulosa cell nuclei

Mannose-binding sites were detected at the ultrastructural level in nuclei of lizard granulosa cells in situ by means of mannosylated ferritin. When ultrathin sections of material embedded in glycol methacrylate were incubated with mannosylated ferritin, a strong labelling was observed over nucleoli, chromatin and the external leaflet of the nuclear envelope, but no labelling was detected in the perinuclear space except for nuclear pores. This labelling was clearly inhibited when sections were incubated in a solution containing both mannosylated ferritin and a sugar-related neoglycoprotein (mannosylated serum albumin). These ultrastructural data supporting the existence of nuclear lectin-like components in reptilian cells are in agreement with our previous findings about such components in nuclei isolated from mammalian cells. Owing to the unique organization of the granular component of the nucleoli in specialized granulosa cells, we are able to show that some of the mannose-binding sites are associated with ribosomal precursors.     

In vitro osteogenic differentiation of human ES cells

Since their isolation in 1998, human embryonic stem (hES) cells have been shown to be capable of adopting various cell fates in vitro. Here, we present in vitro data demonstrating the directed commitment of human embryonic stem cells to the osteogenic lineage. Human ES cells are shown to respond to factors that promote osteogenesis, leading to activation of the osteogenic markers osteocalcin, parathyroid hormone receptor, bone sialoprotein, osteopontin, cbfa1, and collagen 1. Moreover, the mineralized nodules obtained are composed of hydroxyapatite, further establishing the similarity of osteoblasts in culture to bone. These results show that osteoblasts can be derived from human ES cultures in vitro and provide the basis for comparison of adult and embryonic-derived osteogenesis, and for an investigation of potential applications for hES cells in orthopaedic tissue repair.     

Evidence for autocrine activation of a tyrosine kinase in a human gastric carcinoma cell line

Phosphotyrosine (P-Tyr) antibodies have been used to identify the phosphorylated forms of growth factor receptors and oncogene-coded tyrosine kinases. Western blot analysis of a gastric carcinoma cell line with P-Tyr antibodies revealed a tyrosine-phosphorylated protein of Mr 145,000 (P145). In addition, in vitro phosphorylation with (gamma-32P)ATP or P-Tyr immunoprecipitates of the same cells resulted in labelling of this protein on tyrosine. P145 appears to be a transmembrane glycoprotein, with features suggestive of a growth factor receptor. However, the in vivo or in vitro addition of known growth factors did not affect P145 tyrosine phosphorylation. We now report that P145 is rapidly dephosphorylated in vivo when cells are exposed to low pH, a condition known to dissociate ligands from their receptors. The addition of serum-free medium, conditioned by the gastric carcinoma cells, fully restores the tyrosine phosphorylation lost with acid treatment. These data suggest that the activity responsible for P145 phosphorylation on tyrosine, whether intrinsic to P145 itself or due to an associated kinase, is stimulated by a factor secreted by the tumor cells themselves.