Values of Growth Fraction and Cell Loss Factor of Sixty-Five Tumours Re-Examined

Abstract. It has been shown that the mathematical evaluation of data from many cell kinetic experiments, published up to 1977, was rather inaccurate. the discrepancies between published values of growth fraction ( GF ) and the cell loss factor ø, and the results of new calculations based on the same values of LI, T_d, T_c, T_G1, T_s and T_G2, are sometimes surprising. Using a suitable approximation of e^x - 1, the known formulae for GF and ø are replaced by simpler ones of sufficient accuracy. This may help to avoid computational errors in future cell kinetic studies of tumours.     

Kinetics of changes in peritoneal cell populations following acute inflammation

The kinetics of macrophage (M phi) recruitment to the peritoneum following the induction of acute inflammation by thioglycollate broth (TG) was evaluated after prelabeling resident M phi with the fluorescent cell tracking dye, PKH-1. Most of the PKH-1-labeled resident M phi disappeared from the recoverable peritoneal cell population within the first hour after injection of TG. This disappearance coincided with the inflammatory influx of neutrophils (PMNs) and was sustained for at least 5 days after administration of TG, although the PMN number had returned to resident levels by this time. PKH-1-labeled peritoneal M phi were observed again in most animals at 7 days after injection of TG. The number of labeled M phi recovered at 7 days was approximately twice the number of resident peritoneal M phi in control animals which did not receive the TG broth. These additional M phi may include progeny of either the resident M phi or other local M phi precursors, such as omental M phi, which were labeled by the PKH-1 injection.     

Dynamics of cell kinetic parameters during 9L spheroid growth

Cell population kinetics were followed in 9L tumour spheroids as they grew from aggregates of about 80 micron in diameter to over 800 micron. The kinetic parameters measured were cell cycle time, spheroid-doubling time, and growth fraction; from these the cell loss factor phi was calculated. The rate of cell shedding from the surface was also measured, so that the contribution of shedding to the overall cell loss could be evaluated. The major findings include significant elongation of the cell cycle, a low rate of cell death in spheroids below 500 micron in diameter, and a relatively high GF in large spheroids. The results also indicated that 9L spheroid kinetic parameters may be strongly influenced by the culture methodology.     

Flow cytometric evaluation of cell cycle characteristics during in vitro differentiation of chick embryo chondrocytes

The cell cycle kinetic characteristics of chick endochondral chondrocytes differentiating in vitro were studied by flow cytometry. In addition, the synthesis of type I and type X collagens of the same cells was evaluated by immunoprecipitation. Dedifferentiated cells, derived from chick embryo tibiae and grown attached to a substratum, were characterized by type I collagen synthesis, a high growth fraction (GF = 0.94), minimal cell loss factor (phi = 0.02), and a total cell cycle time of the proliferating cells of about 17 h (tG1 = 8 h, tS = 5 h, and tG2 + M = 4 h). Transfer of dedifferentiated cells to suspension culture on agarose-coated dishes induced differentiation to hypertrophic chondrocytes. These were characterized by type X collagen synthesis, a low growth fraction (GF = 0.52), maximal cell loss factor (phi = 1.0), and a total cell cycle time of the proliferating cells of about 73 h (tG1 = 53 h, tS = 12 h, and tG2 + M = 8 h). The transition from dedifferentiated chondrocytes to hypertrophic chondrocytes was accompanied by large increases of the duration of all the cell cycle phases and of the number of quiescent and degenerating cells. Associated with these alterations in cell cycle kinetics was a switch from type I to type X collagen synthesis. Further preliminary data suggest that the population of differentiating chondrocytes (a state between dedifferentiated and hypertrophic chondrocytes) comprises a heterogeneous population of fast and slow growing cells.     

Dynamics of cytochemically distinct subpopulations of macrophages in elicited rat peritoneal exudates

Qualitative and quantitative changes in rat peritoneal macrophage (M phi) subpopulations, differing in their ultrastructural peroxidatic staining characteristics were followed over the course of a thioglycollate (TG) broth-induced inflammatory response. In addition, selected functional features of the normal steady-state and 4-day TG-induced populations of M phi were compared. The steady-state population consisted primarily of M phi with peroxidatic staining limited to the nuclear envelope (NE) and rough endoplasmic reticulum (RER); such cells are called resident M phi. Within hours of TG injection, there was an influx of monocyte-derived exudate M phi, the number of which reached a maximum, by 24 hr. During the next 24 hr, the proportion of exudate M phi decreased with a concomitant increase in peroxidatic activity (PA)-negative M phi. These two cell types continued to predominate for the next 48 hr during which there was a gradual increase in resident M phi and so-called "exudate-resident" M phi, the latter of which exhibits both exudate and resident PA patterns. Thus, the 4-day TG-induced population consisted of four cytochemically distinct M phi subpopulations: approximately 50% PA-negative M phi, approximately 25% exudate M phi, approximately 15% resident M phi, and approximately 10% exudate-resident M phi. Differences in Fc receptors and complement receptors 1 and 3 were noted between the two populations in the presence of progenitors that give rise to colonies of M phi in liquid culture in response to murine-derived colony-stimulating factor 1. The implications of these results in regard to the origin(s) of M phi diversity are discussed.     

Comparative studies of some functional and morphological parameters in the livers of germfree, conventional and ex-germfree mice.

Some parameters of hepatic function and morphology were studied to compare germfree (GF) and conventional (CV) BALB/c mice. The levels of lipid peroxide (LPO) and aniline-hydroxylase (AH) activity in the livers and the serum total cholesterol (TC), triglyceride (TG) and phospholipid (PL) were significantly lower in GF than in CV 8-week-old mice. There were no significant differences in the histology and lectin-histochemistry of the livers in the GF and CV mice. On the other hand, in ex-GF mice which were induced by housing 4-week-old GF mice together with age-matched CV mice, the levels of LPO and AH activity in the liver and the serum TC, TG and PL contents increased rapidly within the first week and then approached values almost identical to those in CV mice 4 weeks later (i.e. at 8 weeks of age). The histologic picture of the liver was similar among the GF, CV and ex-GF mice.     

Endocytosis of growth factor receptors

Binding of a growth factor (GF) to its specific receptor on the cell surface causes the initiation of a signal transduction cascade which eventually results in mitosis. GF:receptor complexes are removed from the cell surface via receptor-mediated endocytosis, a process which involves clathrin-coated pits. After internalization into the endosomal compartment, a significant pool of GFs and GF receptors escape recycling to the cell surface and are sorted to the degradation pathway. The ligandinduced internalization and lysosomal degradation of GF receptors result in the dramatic loss of surface receptors, a phenomenon termed receptor down-regulation. In this review, we discuss relevant biochemical, morphological and kinetic studies of the mechanism of GF endocytosis, and the possible role of this process in mitogenic signaling by growth factor receptors.     

Generation of macrophage cell line from fresh bone marrow cells with a myc/raf recombinant retrovirus

We have studied the effects of infection of fresh murine bone marrow (BM) cells by recombinant retroviruses carrying v-raf and v-myc oncogenes, either alone or in combination. Viruses containing v-raf or v-myc alone failed to induce BM proliferation in 24 out of 27 experiments performed so far, only the J2 virus containing both v-raf and v-myc oncogenes induced BM proliferation. Exogenous growth factors (GF) were not required to sustain the mitogenic effect of J2 virus. Infection with retroviruses carrying only v-raf or v-myc did not induce BM cell growth, indicating that co-expression of the two oncogenes was needed to provide the mitogenic signal(s) for BM proliferation. The kinetics of growth of the J2 virus-infected cells (J2 cells) were characteristically biphasic. The initial burst of proliferation was always followed by a quiescent phase culminating in cell death, which could not be reversed by addition of exogenous GF. In contrast, active proliferation of the quiescent monolayers could be restored by addition of dextran-based beads to the cultures, showing that the growth arrest of J2 cells was a reversible process. J2 cells actively growing in the presence of CT-beads could be expanded and cloned and subsequently grew continuously independent of the CT-beads. Eighteen clones obtained from different infections were all macrophages (M phi) by morphological criteria and all of them expressed the same membrane phenotype compatible with M phi, demonstrating that J2 virus infection leads to immortalization of the same BM-derived monocytic subpopulation. When injected in vivo, J2 cells produced histiocytic tumors in nude mice, but did not grow in immunocompetent syngeneic mice. The cells induced to proliferate in vitro in response to J2 virus infection appeared to be limited to the BM compartment, since spleen cells, thymocytes, peritoneal M phi and liver large granular lymphocytes did not grow in vitro in response to J2 virus. The immortalization of BM cells by J2 virus infection represents a novel reproducible experimental system to deliberately generate M phi lines, which proliferate in response to viral oncogenes and do not require exogenous GF to initiate or to sustain their continuous proliferation.     

Analysis of the growth kinetics of a human lymphoma cell line

The growth kinetics of an established human lymphoma cell line were analyzed by a variety of techniques utilizing various cell inocula (5 X 10(4)--5 X 10(5) cells) dispensed into 60 mm diameter dishes. Techniques included pulse-labeled mitosis (PLM), continuous labeling with 3H-TdR, time-lapse photography (TLP), cell counts by electronic particle counter, and DNA histography obtained by pulse cytophotometry (PCP). There were no significant differences among values determined for any kinetic parameters as a function of cell concentration. The average doubling time of exponentially growing cells, regardless of cell inoculum, was 44.1 hr. The generation time determined by PLM was 31.1 hr with a SD of 4.7 hrs. Transit times for each stage were: TG1 = 10.6 hr, TS = 9.9 hr, TG2 = 9.9 hr, and TM = 0.7 hr. Repeated experiments using continuous labeling with 3H-TdR demonstrated a TG2 of 6.3 hr. The longer value determined by PLM is possibly due to the technical manipulations of this procedure which may delay pulse-labeled cells from resuming cell cycle transit. Hence, values for cell cycle stages were recalculated to give TG1 = 14.1 hr, TS = 9.9 hr, TG2 = 6.3 hr, and TM = 0.7 hr. These results were used to compute the size of each cell cycle stage compartment pool and corresponded very closely to values defined directly by PCP. TLP analysis considered only cells that produced colonies of at least thirty-two cells. Generation times ranged from 8 to 89 hr and showed a positive skewness. The average value measured for 330 divisions was 34.5 hr with a SD of 13.2 hr. Thus, the variance predicted by curve fitting of the PLM data did not correlate with that defined by time-lapse photography nor did it encompass the range in generation times observed directly by TLP. There was a positive correlation between sister-sister cell generation times (+0.66) but no relation was noted for mother-daughter values.     

Fibroblast-secreted macrophage colony-stimulating factor is responsible for generation of biphenotypic B/macrophage cells from a subset of mouse B lymphocytes.

Normal and malignant CD5+ B lymphocytes can develop macrophage-like characteristics. One stimulus of this phenotypic shift is culture of normal mouse splenic B lymphocytes with splenic fibroblasts or their conditioned media. These biphenotypic B/macrophage (B/M phi) cells simultaneously display macrophage characteristics, such as phagocytosis and F4/80 expression, while retaining B cell features, including expression of surface Ig, CD5, B220, and rearranged Ig genes. The present study investigated the fibroblast-secreted factor that promotes this phenotypic change from B cell to B/M phi cell. RT-PCR analysis demonstrated that mRNA for M-CSF is produced by splenic fibroblasts. Recombinant M-CSF (CSF-1) could replace fibroblast-conditioned medium to elicit the development and survival of B/M phi cells from splenic B lymphocytes. In addition, neutralization of fibroblast-secreted M-CSF with specific mAbs abrogated the ability of conditioned supernatants to promote outgrowth of B/M phi cells. The transition from B lymphocyte to B/M phi cell was marked by the kinetic appearance of mRNA for the M-CSF receptor, c-fms, at day 3 following culture initiation. These results demonstrate that M-CSF is important in the development and physiology of mouse B/M phi cells and potentially in the growth of human biphenotypic hematological malignancies. Interestingly, the presence of IFN-gamma in splenic B lymphocyte cultures abrogated the effect of fibroblast-conditioned medium or M-CSF on outgrowth of B/M phi cells. Furthermore, these findings suggest that a Th1 microenvironment favored by typical macrophages is detrimental to the outgrowth of B/M phi cells.     

Initiation of transcription by T7 RNA polymerase as its natural promoters.

A kinetic assay has been developed to measure the strength of natural T7 promoters. By determining the rate of appearance of initiation products in the presence of constant concentrations of T7 RNA polymerase, an incomplete mixture of ribonucleoside triphosphates, and increasing promoter concentrations, a maximum rate of product formation (Vmax) and a promoter concentration giving half of the maximal activity ([P]Vmax/2) can be determined for any cloned T7 promoter. On supercoiled plasmids, it was found that the [P]Vmax/2 measured for the six promoters phi 1.1B, phi 1.3, phi 3.8, phi 6.5, phi 10, and phi 13 ranged from 3.4 +/- 1.1 to 12.0 +/- 2.4 nM while the Vmax values showed no significant trends. On plasmids that had been linearized by cleavage at a single site with a restriction endonuclease, the cloned T7 promoters assayed fell into two broad classes that appear to be characterized by the T7 class II and III promoters. Generally, the class II promoters required higher promoter concentrations to produce half of the maximum rates of initiation ([P]Vmax/2 values) than the class III promoters. The [P]Vmax/2 values for the class II promoters ranged from 20 +/- 2.7 to 23 +/- 3.6 nM, while the [P]Vmax/2 values for the class III promoters phi 10 and phi 13 were 13 +/- 1.6 nM and 7.8 +/- 1.4 nM. The one exception is the class III promoter phi 6.5 whose [P] Vmax/2 (17 +/- 5 nM) falls between the [P]Vmax/2 values of the class II promoters and the strong class III promoters. The Vmax values measured on linear templates are variable, but it appears that phi 10 is more active than the other five promoters.     

Transport Inhibition of Digoxin Using Several Common P-gp Expressing Cell Lines Is Not Necessarily Reporting Only on Inhibitor Binding to P-gp

We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918) to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute) confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC₅₀ measurements using digoxin as a probe substrate in this cell line could be due to inhibition of P-gp, of digoxin uptake transport, or both. This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH (National Institute of Health), Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown). These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1) bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxin''s cellular uptake; (2) partition into the basolateral membrane and directly reduce membrane permeability; (3) aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while highly permeable P-gp substrates such as amprenavir, quinidine, ketoconazole and verapamil do not, regardless of whether they actually use the basolateral transporter.     

Interleukin-2-activated murine cell lines with macrophage- and B-lymphoblast-lytic activity.

Interleukin-2 (IL-2)-activated murine killer cell lines with macrophage- and B-lymphoblastic-lytic activity were established, and their target specificity, surface markers, recognition-related structures, and requirements for optimal cell growth were characterized. Sustained growth of IL-2-activated lymphocytes was supported by the combination of IL-2 and IL-4-enriched T cell conditioned medium (CM), but was not supported by IL-2 alone or the combination of IL-2 and IL-3-containing CM in the presence of macrophages (M phi). The established line required continuous contact with M phi to maintain anti-M phi cytolytic activity. Flow cytometric analysis showed that the original line isolated by the first cloning was Thyl+, CD4-, and weakly CD8+, FcR+. The majority of these cells were CD3+ and TCR-V beta 8+. From this line, the CD3+, TCR-V beta 8+ and CD3-, TCR-V beta 8- clones were isolated by subcloning. The former clone showed Thyl+, CD3+, CD4-, CD8-, TCR-V beta 8+, FcR(+)-phenotype, and the latter clone showed Thyl+, CD3-, CD4-, CD8-, TCR-V beta 8-, FcR- phenotype. The original line and subclones showed a similar target specificity and killed resident or thioglycollate (TG)-induced peritoneal M phi and B-lymphoblasts, but did not kill T-lymphoblasts. Allogeneic M phi, M phi-like cell line P388D1, and B cell hybridoma were sensitive, whereas fresh lymphocytes, T cell lymphoma BW5147, natural killer (NK)-sensitive YAC-1, and NK-resistant P815 tumor cells were resistant to lysis by these cytotoxic lines. The addition of anti-H-2 heteroserum, anti-MHC class 1, anti-MHC class II, anti-CD3, or anti-TCR-V beta 8 monoclonal antibody (mAb) to assay cultures did not inhibit the anti-M phi cytolysis by these killer cells. In addition, the CD3- TCR-V beta 8- clone killed M phi and B lymphoblasts better than the CD3+, TCR-V beta 8+ clone. These results suggest that cytotoxic lines established in this study do not use the T cell receptor (TCR) molecules to recognize target cells and the MHC molecules are not involved in recognition. Anti-LFA-1 mAb partially inhibited anti-M phi-lysis, suggesting that the cell contact between targets and effectors is important in cytolysis. Our present data suggest that the culture condition containing IL-2, IL-4, and M phi may support the continuous growth of non-MHC-restricted killer cells with relative target specificity against M phi and B-lymphoblasts.     

Cytotoxic ether phospholipids. Different affinities to lysophosphocholine acyltransferases in sensitive and resistant cells

Alkyllysophospholipids (ALP) which are 1-O-alkyl analogs of the cell membrane component 1-acyl-sn-glycero-3-phosphocholine (1-acyl-GPC) represent a family of new antitumor drugs. Susceptibility of cells to ALP is correlated to a selective inhibition of fatty acid incorporation into 1,2-diacyl-sn-glycero-3-phosphocholine in intact cells. This report examines oleoyl-CoA-1-acyl-GPC acyl-transferase activities in cell-free systems of ALP-sensitive methylcholanthrene-induced fibrosarcoma cells (MethA cells) and ALP-resistant bone marrow-derived murine macrophages (BMM phi). The specific activities for the oleoyl-CoA-1-acyl-GPC acyltransferases were 1.05 +/- 0.06 nmol X mg-1 X min-1 and 2.98 +/- 0.27 nmol X mg-1 X min-1, respectively. The kinetic parameters for 1-palmitoyl-GPC were Km = 16.6 microM, Vmax = 4.3 nmol X mg-1 X min-1 (BMM phi) and Km = 7.6 microM, Vmax = 2.0 nmol X mg-1 X min-1 (MethA cells). In the presence of 1-O-octadecyl-2-O-methyl racemic glycero-3-phosphocholine (ET-18-OCH3), one of the most potent cytotoxic ALP, the acyltransferase was dose dependently inhibited in MethA cells with a 50% inhibition concentration at 40 micrograms/ml. The BMM phi-acyltransferase was not affected up to 80 micrograms of ET-18-OCH3/ml. The kinetic parameters (Km' = 15.4 microM, Vmax' = 2.2 nmol X mg-1 X min-1) suggest that ET-18-OCH3 is a competitive inhibitor in MethA cells. Inhibitor constants for ET-18-OCH3, calculated from Dixon plots, were found to be 423 microM (BMM phi) and 13 microM (MethA cells) indicating a 33-fold larger affinity of ET-18-OCH3 to the MethA cells than to the BMM phi acyltransferase. From these data we assume that the inhibition of oleic acid incorporation into cellular phosphocholine during the antineoplastic action of ALP may be due to different affinities of the inhibitor to the 1-acyl-GPC acyltransferases in different cell types.     

Studies on the population kinetics of the Walker carcinoma by autoradiography and pulse cytophotometry.

The proliferation parameters of the Walker carcinoma were estimated from both in vivo and in vitro measurements. tthe transplantable Walker carcinoma 256 was grown in male inbred BD1 rats. During exponential growth, 5--6 days after transplantation, a PLM curve was performed, yielding estimates of TC approximately equal to 18-0 hr, TS approximately equal to 6-4 hr, TG2+M approximately equal to 4-1 hr. With the double labelling technique in vitro under 2-2 atm oxygen we obtained: TC approximately equal to 18-2 hr, TS approximately equal to 8-2 hr, TG2+M approximately equal to 2-0 hr. From pulse cytophotometry DNA content histograms the fractions of cells in the cell cycle phases were calculated using a computer program: fG1 approximately equal to (47-6 +/- 1-1)%, fS approximately equal to (34-1 +/- 1-0)%, fG2+M approximately equal to (18-3 +/- 1-5)%. These fractions remained constant between the fifth and the twelfth day after transplantation. At that time the tumour growth had already slowed down appreciably. The growth fraction determined by repetitive labelling was 0.96 on the fifth and 0-93 on the seventh and eleventh day. The cell loss factor was phi approximately equal to 17% during exponential tumor growth and increased to about 100% between the tenth and twelfth day. The agreement of the cell kinetic data determined by autoradiography from solid tumours in vivo (PLM, continuous labelling) and autoradiography as well as pulse cytophotometry from in vitro experiments (excised material) was satisfactory.     

Binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase, bovine alpha-chymotrypsin and subtilisin Carlsberg: thermodynamic study

The effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the recombinant proteinase inhibitor eglin c from leech Hirudo medicinalis to human leukocyte elastase (EC 3.4.21.37), bovine alpha-chymotrypsin (EC 3.4.21.1) and subtilisin Carlsberg (EC 3.4.21.14) has been investigated. On lowering the pH from 9.5 to 4.5, values of Ka for eglin c binding to the serine proteinases considered decrease thus reflecting the acid-pK shift of the invariant histidyl catalytic residue (His57 in human leukocyte elastase and bovine alpha-chymotrypsin, and His64 in subtilisin Carlsberg) from congruent to 6.9, in the free enzymes, to congruent to 5.1, in the enzyme:inhibitor adducts. At pH 8.0, values of the apparent thermodynamic parameters for eglin c binding are: human leukocyte elastase - Ka = 1.0 x 10(10) M-1, delta G phi = -13.4 kcal/mol, delta H phi = +1.8 kcal/mol, and delta S phi = +52 entropy units; bovine alpha-chymotrypsin -Ka = 5.0 x 10(9) M-1, delta G phi = -13.0 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units; and subtilisin Carlsberg - Ka = 6.6 x 10(9) M-1, delta G phi = -13.1 kcal/mol, delta H phi = +2.0 kcal/mol, and delta S phi = +51 entropy units (values of Ka, delta G phi and delta S phi were obtained at 21 degrees C; values of delta H phi were temperature independent over the range explored, i.e. between 10 degrees C and 40 degrees C; 1 kcal = 4184J).(ABSTRACT TRUNCATED AT 250 WORDS)     

Zinc Deficiency Induced in Swiss 3T3 Cells by a Low-Zinc Medium Impairs Calcium Entry and Two Mechanisms of Entry Are Involved

Zinc deficiency in 3T3 cells induced by the use of diethylenetriaminepentaacetate (DTPA) has been shown to impair calcium entry associated with failure of proliferation when the cells are stimulated with polypeptide growth factors (GF). These functions of zinc have been evaluated here in the same clone of cells by simple depletion using a low-zinc medium (0.05 μmol/L zinc) without chelator. Confluent cells were maintained for 1 day in the low-zinc medium without GF, then loaded with Fluo-4, and stimulated with GF. Calcium entry was measured by the increase in sustained fluorescence. It was preceded by the release of stored calcium as observed in the previous study using DTPA. Zinc deprivation decreased calcium entry when calcium was added at 0 or 0.05 mmol/L but not when 0.1 mmol/L or higher. Cell proliferation reflected similar effects of zinc and calcium concentrations. In a newly acquired clone of 3T3 cells, GF did not induce internal calcium release but thapsigargin (TG) did. When added in a low-calcium medium, both agonists stimulated calcium entry when external calcium was added, suggesting that two different mechanisms of entry were impaired by zinc deficiency. Zinc deficiency produced by DTPA in the newer clones gave similar results, decreasing calcium entry induced by both agonists. The effects of GF and TG were not additive. The results confirm the earlier observation that zinc deficiency impairs calcium entry into 3T3 cells when stimulated by GF and show that the cells can take up calcium by either store-operated or receptor-operated mechanisms.     

Bronchoalveolar lavage fluid differential cell count. How many cells should be counted?

OBJECTIVE: To investigate the number of cells to be counted in cytocentrifuged bronchoalveolar lavage (BAL) fluid preparations in order to reach a reliable enumeration of each cell type. STUDY DESIGN: A total of 136 BAL fluid samples for patients with suspected pneumonia or interstitial lung disease were investigated. Differential cell counts were performed on May-Grünwald-Giemsa-stained cytocentrifuged preparations by 2 observers, each differentiating 500 cells. Reliability for the enumeration of each cell type was expressed as phi value, as calculated in generalizability theory. RESULTS: For polymorphonuclear neutrophils (PMNs), alveolar macrophages, lymphocytes and eosinophils, an acceptable phi value of > or = .95 was reached at a count of 300 cells by 1 observer. Mast cells reached a phi value of only .674 at a count of 500 cells by 1 observer, precluding a reliable count. At a count of 500 cells by 1 observer, squamous epithelial cells, bronchial epithelial cells and plasma cells displayed phi values of .868, .903 and .816, respectively. CONCLUSION: At a count of 300 cells, PMNs, alveolar macrophages, lymphocytes and eosinophils are reliably enumerated in cytocentrifuged BAL fluid samples.     

Cell cycle kinetics of uninfected and feline leukemia virus-infected canine lymphoma cell lines: effects of methotrexate treatment

The cell cycle kinetics of uninfected and feline leukemia virus-infected canine lymphoma cell lines were determined by autoradiography (PLM method) as follows: DT-5: generation time (TC), 15.2 h; pre-synthetic gap phase (TG1), 3.2 h; DNA-synthetic phase (TS), 8.2 h; post-synthetic gap ph se (TG2), 3.3 h; visible mitotic phase (TM), 0.5 h. 11028: TC, 13.6 h; TG1, 1.9 h; TS, 7.7 h; TG2, 3.4 h; TM, 0.6 h. 11028+FeLV (11028 productively infected with feline leukemia virus): TC, 11.2 h; TG1, 0.2 h; TS, 8.3 h; TG2, 2.1 h; TM, 0.6 h. Exposure of the lymphoma cell lines to methotrexate (MTX) in vitro produces dose-related increases in cellular volume, associated with reductions in cellular proliferation. The relative sensitivities of these cell lines to MTX, measured by the ID50 MTX concentrations for DT-5, 11028, and 11028+FeLV are 118 nM, 122 nM, and 28 nM respectively. The cell kinetic effects of the ID50 MTX concentrations added to cultures of lymphoma cells pulse-labeled with tritiated thymidine are an approximately 2-h prolongation of TC, attributable to a lengthening of TS, with other cell cycle phases not significantly altered. These cell lines are highly tumorigenic when transplanted into the cheek pouches of immunosuppressed hamsters, with inocula of 10(4) cells producing rapidly growing, well vascularized tumors.