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E A Musgrove

Publications and source records attributed to E A Musgrove.

At least 55 records · Page 3Linked to original sources
PubMed1993

Acute effects of growth factors on T-47D breast cancer cell cycle progression.

Growth factors play a major role in the control of human breast cancer cell proliferation but their acute effects on cell cycle progression have not been well studied in these cells. T-47D cells, growth-inhibited by serum deprivation, were induced to re-enter the cell cycle in a concentration- and time-dependent manner by addition of insulin, insulin-like growth factor (IGF)-I, epidermal growth factor (EGF), transforming growth factor alpha (TGF alpha) or basic fibroblast growth factor (bFGF). After a lag of approximately 10 h semi-synchronous entry into S phase was observed. The relative potencies of maximal concentrations of growth factors were in the order: insulin approximately IGF-I approximately bFGF > TGF alpha > EGF, identifying bFGF as among the most potent mitogens for these cells. Insulin or IGF-I alone resulted in growth rates comparable with those observed in fetal calf serum. These data demonstrate that single growth factors can induce a significant proportion of T-47D cells to traverse the cell cycle. The kinetics for entry into S phase were similar, indicating that the basis of differential sensitivity to the growth factors tested was the proportion of cells that responded and ultimately entered S phase.

Breast Neoplasms↗
PubMed1993

Cyclin gene expression and growth control in normal and neoplastic human breast epithelium.

Recent advances in defining the molecular mechanisms of cell cycle control in eukaryotes provide a basis for better understanding the hormonal control of cell proliferation in normal and neoplastic breast epithelium. It is now clear that a number of critical steps in cell cycle progression are controlled by families of serine/threonine kinases, the cdks. These kinases are activated by interactions with various cyclin gene products which form the regulatory subunits of the kinase complexes. Several families of cyclins control cell cycle progression in G1 phase, cyclins C, D and E, or in S, G2 and mitosis, cyclins A and B. Recent studies have defined the expression and regulation of cyclin genes in normal breast epithelial cells and in breast cancer cell lines. Following growth arrest of T-47D breast cancer cells by serum deprivation restimulation with insulin results in sequential induction of cyclin genes. Cyclin D1 mRNA increases within 1 h of mitogenic stimulation and is followed by increased expression of cyclins D3 and E in G1 phase, cyclin A in late G1/early S phase and cyclin B1 in G2. Similar results were observed following epidermal growth factor stimulation of normal breast epithelial cells. Other hormones--oestrogens and progestins--and growth factors--insulin-like growth factor-I and basic fibroblast growth factor--with actions in G1 were also investigated for their effects on G1 cyclin gene expression. In all cases there was an excellent correlation between the induction of cyclin D1 mRNA and subsequent entry into S phase. Furthermore, growth inhibition by antioestrogens and concurrent G1 arrest were preceded by an acute decrease in cyclin D1 gene expression. These observations suggest a likely role for cyclin D1 in mediating many of the known hormonal effects on cell proliferation in breast epithelial cells.

Breast↗
PubMed1993

Growth factor, steroid, and steroid antagonist regulation of cyclin gene expression associated with changes in T-47D human breast cancer cell cycle progression.

Cyclins and proto-oncogenes including c-myc have been implicated in eukaryotic cell cycle control. The role of cyclins in steroidal regulation of cell proliferation is unknown, but a role for c-myc has been suggested. This study investigated the relationship between regulation of T-47D breast cancer cell cycle progression, particularly by steroids and their antagonists, and changes in the levels of expression of these genes. Sequential induction of cyclins D1 (early G1 phase), D3, E, A (late G1-early S phase), and B1 (G2 phase) was observed following insulin stimulation of cell cycle progression in serum-free medium. Transient acceleration of G1-phase cells by progestin was also accompanied by rapid induction of cyclin D1, apparent within 2 h. This early induction of cyclin D1 and the ability of delayed administration of antiprogestin to antagonize progestin-induced increases in both cyclin D1 mRNA and the proportion of cells in S phase support a central role for cyclin D1 in mediating the mitogenic response in T-47D cells. Compatible with this hypothesis, antiestrogen treatment reduced the expression of cyclin D1 approximately 8 h before changes in cell cycle phase distribution accompanying growth inhibition. In the absence of progestin, antiprogestin treatment inhibited T-47D cell cycle progression but in contrast did not decrease cyclin D1 expression. Thus, changes in cyclin D1 gene expression are often, but not invariably, associated with changes in the rate of T-47D breast cancer cell cycle progression. However, both antiestrogen and antiprogestin depleted c-myc mRNA by > 80% within 2 h. These data suggest the involvement of both cyclin D1 and c-myc in the steroidal control of breast cancer cell cycle progression.

Breast Neoplasms↗
PubMed1993

Expression and amplification of cyclin genes in human breast cancer.

Cyclins, the regulatory subunits of cyclin-dependent kinases, play an important role in the control of cellular proliferation. Since dysregulated expression of these genes may contribute to the malignant phenotype the expression and amplification of cyclin A, B1, C, D1, D2, D3 and E genes were studied in 20 breast cancer cell lines. Increased expression of one or more of the cyclin A, B1, D1 or E genes was found in seven cell lines (35%); of these five (25%) showed increased expression of cyclin D1. Overexpression occurred in both the presence and absence of gene amplification. Conversely, amplification did not invariably lead to overexpression. Cyclin D2 expression was lower in breast cancer cell lines than in cultured normal breast epithelial cells. Cyclin D1 expression was further investigated in breast tumour biopsies: 56 of 124 specimens (45%) expressed higher levels of cyclin D1 mRNA than normal breast tissue. These data implicate dysregulated expression of several cyclin genes, particularly cyclin D1, as a potential factor in the pathogenesis of breast cancer.

Base Sequence↗
PubMed1992

Regulation of breast cancer cell cycle progression by growth factors, steroids and steroid antagonists.

The control of human breast cancer cell proliferation in vitro is known to involve complex interactions between steroid hormones, peptide hormones and growth factors. Little is known, however, of the mechanisms by which these factors, alone or in combination, control cell cycle progression and the expression of specific genes involved in cell cycle control. A pre-requisite for such studies is a cellular system in which non-proliferating or slowly proliferating cells can be maintained in a defined environment and stimulated to progress through the cell cycle by addition of hormones and growth factors. Such a system has been developed for T-47D human breast cancer cells: quiescent or slowly proliferating cells maintained in a serum-free medium can be stimulated to increase their rate of cell cycle progression upon a single addition of insulin, IGF-I, EGF, TGF alpha or bFGF. Oestradiol alone was ineffective but caused a significant increase in % S phase cells when added in the presence of insulin. Progestins, in the presence of absence of insulin, had a biphasic effect with an initial increase in cell cycle progression followed by cell cycle arrest. Both antioestrogens and the antiprogestin, RU 486, in the absence of oestrogen or progestin, were potent inhibitors of insulin-induced proliferation. Increases in cell cycle progression were invariably accompanied by acute increases in c-fos and c-myc mRNA levels. Induction of c-myc by oestrogen and progestin was inhibited by antioestrogens and RU 486, respectively. These data illustrate that the culture of breast cancer cells in a serum-free, chemically defined environment provides an excellent model in which to define the role of individual factors involved in breast cancer growth control. The biological data derived from this system provide a basis for identifying and characterizing genes involved in the control of cell cycle progression in human breast cancer.

Breast Neoplasms↗
PubMed1991

Progestins both stimulate and inhibit breast cancer cell cycle progression while increasing expression of transforming growth factor alpha, epidermal growth factor receptor, c-fos, and c-myc genes.

This study documents a biphasic change in the rate of cell cycle progression and proliferation of T-47D human breast cancer cells treated with synthetic progestins, consisting of an initial transient acceleration in transit through G1, followed by cell cycle arrest and growth inhibition. Both components of the response were mediated via the progesterone receptor. The data are consistent with a model in which the action of progestins is to accelerate cells already progressing through G1, which are then arrested early in G1 after completing a round of replication, as are cells initially in other phases of the cell cycle. Such acceleration implies that progestins act on genes or gene products which are rate limiting for cell cycle progression. Increased production of epidermal growth factor and transforming growth factor alpha, putative autocrine growth factors in breast cancer cells, does not appear to account for the initial response to progestins, since although the mRNA abundance for these growth factors is rapidly induced by progestins, cells treated with epidermal growth factor or transforming growth factor alpha did not enter S phase until 5 to 6 h later than those stimulated by progestin. The proto-oncogenes c-fos and c-myc were rapidly but transiently induced by progestin treatment, paralleling the well-known response of these genes to mitogenic signals in other cell types. The progestin antagonist RU 486 inhibited progestin regulation of both cell cycle progression and c-myc expression, suggesting that this proto-oncogene may participate in growth modulation by progestins.

Blotting, Northern↗
PubMed1990

Differential effects of phorbol ester on epidermal growth factor receptors in estrogen receptor-positive and -negative breast cancer cell lines.

A previous study from this laboratory (Koga et al., Cancer Res., 48: 2734-2739, 1988) demonstrated that the growth inhibitory effect of 1,25-dihydroxyvitamin D3 in human breast cancer cells in vitro was associated with a decline in the concentration of epidermal growth factor receptor (EGF-R). In the present study experiments were undertaken with the phorbol ester, 12-O-tetradecanoyl-phorbol-13-acetate (TPA), an agent known to decrease EGF-R binding, in order to further define the relationship between changes in EGF-R binding and changes in growth rates in 10 human breast cancer cell lines. Treatment with TPA decreased the rate of cell proliferation in all cell lines except BT 474 in which a slight increase in proliferation rate was observed. Sensitivity to TPA was unrelated to estrogen receptor (ER) status and a wide spectrum of sensitivities was apparent. The concentrations of TPA that produced a 25% decrease in cell number ranged from less than 0.25 nM for MCF 7M and BT 20 cells to greater than 10 nM for the HBL 100, T 47D, and ZR 75-1 cell lines. Growth inhibition was associated with a block in cell cycle progression in the G1 and G2 + M phases of the cell cycle. In all cell lines studied, except BT 474, TPA treatment resulted in a reduction in the ability of cells to bind EGF. Saturation analysis revealed marked differences between the effects of TPA on EGF binding in ER+ and ER- cell lines. In ER+ cell lines, 2-h treatment with 10 nM TPA resulted in a marked reduction in the number of high affinity EGF-R sites and a significant decrease in binding affinity. Among this group of cell lines there was a significant positive correlation between the ability of TPA to decrease cell growth and the TPA-induced decrease in the number of EGF-R sites/cell (r = 0.82, P less than 0.03). In ER- cell lines, TPA-induced growth inhibition and the minor changes in EGF-R concentration were unrelated. However, growth inhibition was negatively correlated with TPA-induced changes in apparent affinity of the EGF-R (r = 1.00, P less than 0.003) and in the same rank order as the EGF-R concentration in control cells. These data demonstrate differential relationships between TPA-induced changes in growth and EGF-R binding in ER+ and ER- breast cancer cells, thus supporting the view that growth regulatory pathways are markedly different in these two subtypes of human breast cancer.

Breast Neoplasms↗
PubMed1989

Points of action of estrogen antagonists and a calmodulin antagonist within the MCF-7 human breast cancer cell cycle.

Tamoxifen and other structurally related nonsteroidal antiestrogens possess properties in addition to their estrogen antagonist activity including inhibition of both calmodulin and protein kinase C. The present studies were designed to test whether the estrogen-reversible (estrogen receptor mediated) and estrogen-irreversible effects of nonsteroidal antiestrogens on cell cycle progression in vitro were mediated at the same or different points within the cell cycle and if the estrogen-irreversible effects coincided temporally with that of a calmodulin antagonist, R24571. Initial experiments investigated the effects of ICI 164384, a pure estrogen antagonist, on proliferation kinetics in asynchronous cultures of MCF-7 human breast cancer cells. At concentrations greater than 1 nM ICI 164384 significantly reduced growth rate while at greater than or equal to 50 nM, ICI 164384 completely arrested growth after the first 24 h of exposure. Concentrations up to 5 microM failed either to cause more profound effects on growth or induce cytotoxicity. Growth inhibition was associated with a decrease in the proportion of S phase cells and an accumulation of cells in G1 phase, and was completely reversed by the simultaneous addition of equimolar estradiol. In order to identify the points of action within the cell cycle of ICI 164384, and the estrogen-reversible and estrogen-irreversible components of the nonsteroidal estrogen antagonist, hydroxyclomiphene, and the calmodulin antagonist, R24571, experiments were undertaken with MCF-7 cells synchronized by mitotic selection. The mean point of action was assessed by delaying addition of the drugs for increasing time periods following mitotic selection and using DNA flow cytometry to determine the proportion of the population affected by drug administration at a specific time within G1 phase. These studies showed that sensitivity to ICI 164384 was restricted to the early part of G1 phase and that the mean time of action was 4.9 h after the beginning of G1 for this pure estrogen antagonist. The mean times of action of the estrogen-reversible (4.1 h into G1 phase) and estrogen-irreversible (4.1 h) mechanisms of action of hydroxyclomiphene, and R24571 (4.0 h), all appeared to be within a similar time frame in early to mid G1 phase. It is concluded that ICI 164384 inhibits breast cancer cell proliferation by inducing a transition delay in G1 phase and that the point of action of this pure estrogen antagonist in early G1 phase is indistinguishable temporally from that of nonsteroidal antiestrogens and calmodulin antagonists.

Breast Neoplasms↗
PubMed1989

Modulation of the growth-inhibitory effects of progestins and the antiestrogen hydroxyclomiphene on human breast cancer cells by epidermal growth factor and insulin.

The molecular basis of the growth-inhibitory effects of progestins or antiestrogens in human breast cancer has not been fully elucidated. Both direct actions and indirect actions, where the growth inhibition results from modulation of the production of, and/or the response to, growth factors, have been proposed. In this study the ability of some growth factors to modulate progestin-induced inhibition of cell proliferation was investigated in vitro, using T-47D human breast cancer cells. When T-47D cells grown in insulin-containing medium were treated for 4 to 5 days with the synthetic progestin, ORG 2058, at a concentration of 10 nM, cell numbers were reduced to 10 to 20% of control. Simultaneous treatment with epidermal growth factor (EGF) and ORG 2058 led to a partial reversal of the growth-inhibitory effect of the progestin. The magnitude of the effect of EGF was concentration dependent, being half-maximal at 0.48 ng/ml (0.08 nM) and maximal at concentrations greater than 5 ng/ml (greater than 0.8 nM), where cell numbers were increased by 50% compared to those in the presence of ORG 2058 alone. ORG 2058 was no more potent in the absence of insulin, and, after several passages in insulin-free medium, addition of insulin failed to modulate the effect of ORG 2058. However, when maximal concentrations of insulin (5 micrograms/ml) and EGF (10 ng/ml) were administered together with ORG 2058, insulin and EGF appeared to act synergistically to reduce the ORG 2058-induced inhibition of proliferation. In similar experiments in which cells were treated with hydroxyclomiphene, a potent antiestrogen, insulin was shown to partially reverse the growth-inhibitory effects of hydroxyclomiphene. Significant increases in cell number above hydroxyclomiphene-treated controls were apparent at insulin concentrations greater than 50 ng/ml, and at 5 micrograms/ml the increase was approximately 2-fold. In contrast to the situation with progestins, simultaneous treatment with EGF and insulin had only an additive effect in reversing the growth-inhibitory effect of the antiestrogen. The results are compatible with the hypothesis that part of the growth-inhibitory effects of progestin and antiestrogen on human breast cancer cell proliferation is mediated by inhibition of autocrine growth factor production. However, they do not exclude more direct mechanisms involving modulation of progesterone and/or estrogen receptors by EGF and/or insulin.(ABSTRACT TRUNCATED AT 400 WORDS)

Breast Neoplasms↗
PubMed1989

Effects of 1,25-dihydroxyvitamin D3 on cell-cycle kinetics of T 47D human breast cancer cells.

The replication of several human and animal cancer cell lines is regulated in vitro and in vivo by 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3], the hormonally active form of vitamin D3. We have examined the effects of concentrations of 1,25-(OH)2D3, which inhibit cellular replication, on the cell-cycle kinetics of a 1,25-(OH)2D3-responsive human breast cancer cell line, T 47D. After 6 or 7 days of treatment, a time period representing approximately five cell population doublings of control cultures, concentrations of 1,25-(OH)2D3 in the range 10(-9) M to 10(-6) M caused a time- and concentration-dependent decrease in cell numbers. Treatment of cells growing in charcoal-treated fetal calf serum with 10(-8) M 1,25-(OH)2D3 for 6 days reduced cell numbers to 49% +/- 9% (n = 9) of control, and this was associated with a marked increase in the proportion of cells in the G2 + M phase of the cell cycle from 9.7% +/- 0.5% (n = 11) to 19.6% +/- 2.3% (n = 9), significant by paired analysis (P less than 0.002). At higher concentrations of 1,25-(OH)2D3 (10(-7)-10(-6) M), there was a concentration-dependent decline in S phase and increases in both G0/G1 and G2 + M phase cells. Detailed analysis of the temporal changes in cell-cycle phase distribution following treatment with 2.5 X 10(-8) and 10(-7) M 1,25-(OH)2D3 showed an initial accumulation of cells in G0/G1 and depletion of S phase cells during the first 24 hr of treatment. This decline in S phase cells was not accompanied by a decline in % G2 + M indicating a transition delay in G2 or mitosis. At the lower dose these changes returned to control values at 48 hr and at later times were associated with a slight but consistent decline in G0/G1 phase and an increase in G2 + M. In contrast cells treated with 10(-7) M 1,25-(OH)2D3 had significantly elevated % G0/G1 cells at days 2 and 3, consistent with a transition delay through G1 phase. This was confirmed in stathmokinetic experiments which demonstrated an approximate sevenfold decrease in the rate of exit of cells from G0/G1 following 4 days of exposure to 10(-7) M 1,25-(OH)2D3. This accumulation of cells in G0/G1 was accompanied by a fall in % S phase cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Breast Neoplasms↗
PubMed1989

Enumeration of 6-thioguanine-resistant tumour cells using flow cytometry and comparison with a microtitration cloning assay.

Measurement of mutant frequency in tumour specimens has been hampered by low cloning efficiency in soft agar. A method was developed to detect cell proliferation using the thymidine analogue 5-bromo-2'-deoxyuridine (BrUdR). BrUdR incorporation was monitored by immunofluorescent staining of fixed cells using a monoclonal antibody highly specific for this nucleoside analogue. The 6-thioguanine (6TG) exposure conditions which inhibited DNA synthesis, as measured by BrUdR incorporation, in wild-type cells while allowing proliferation of spontaneous hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutants were investigated using tumour cell lines. It was shown that exposure to 10(-5) M BrUdR for the equivalent of 1 cell cycle time did not affect growth of wild-type cells, nor did it affect the growth of HPRT- mutants in the presence of 6TG. Methods for rapid flow cytometric enumeration of BrUdR-labelled 6TG-resistant cells were developed using fluorescent microspheres as an internal standard. To validate the BrUdR mutation assay, the 6TG mutant frequency (MF) was measured in L1210 R/S, a mouse leukaemic cell line (BrUdR 6TG MF = 7.0 X 10(-5] and the results directly compared with those from a microtitration cloning assay (MF = 4.6 X 10(-5]. The results were similar and within the range reported for HPRT MF in mammalian cells.

Animals↗
PubMed1988

Flow cytometric enumeration of drug-resistant tumor cells.

In an attempt to elucidate the role of somatic mutation in the development of resistance to cancer chemotherapy, an assay was sought to measure the frequency of hypoxanthine-guanine phosphoribosyltransferase (HPRT) mutants in human tumors. Based on the same principle as [3H]thymidine/autoradiography, a method was developed to identify cell proliferation using the thymidine analog 5-bromo-2'-deoxyuridine (BrdUrd). BrdUrd incorporation into DNA was measured following the immunofluorescent staining of fixed cells using a monoclonal antibody highly specific for this nucleoside analog. The human leukemic cell line, CCRF-CEM, was used to investigate the conditions necessary for the stringent selection of HPRT- mutants using 6-thioguanine (6TG). The appropriate 6TG exposure necessary to inhibit BrdUrd incorporation in wild-type cells, while allowing proliferation of spontaneous HPRT- mutants, was greater than or equal to 30 microM 6TG for 72 h (10 microM BrdUrd added 24 h prior to harvest). BrdUrd did not affect the growth of HPRT- mutants in the presence of 6TG. BrdUrd-labeled 6TG-resistant cells were enumerated flow cytometrically using fluorescent microspheres as an internal standard and the nonparametric, Kolmogorov-Smirnov test was used for independent statistical analysis of the subpopulations of fluorescent, 6TG-resistant cells. Evidence that CCRF-CEM cells which incorporated BrdUrd in the presence of 6TG were, in fact, HPRT- mutants was sought. It was demonstrated that spontaneous 6TG-resistant cells from the CCRF-CEM population were reduced by growth in medium containing aminopterin. The mutant frequency in the CCRF-CEM cell line was found to be 4.28 x 10(-5) +/- 2.04 x 10(-5) using the BrdUrd/flow cytometric technique.

Bromodeoxyuridine↗
PubMed1988

Effect of medroxyprogesterone acetate on proliferation and cell cycle kinetics of human mammary carcinoma cells.

The effect of medroxyprogesterone acetate (MPA) on breast cancer cell proliferation kinetics was investigated in ten human breast cell lines growing as monolayer cultures. Significant inhibition of growth occurred only in the estrogen receptor-positive, progesterone receptor-positive cell lines, T-47D, MCF-7, ZR 75-1, BT 474, and MDA-MB-361. Among these cell lines sensitivity to MPA varied widely; concentrations required for 20% inhibition of growth ranged from 0.04 nM for T-47D to greater than 100 nM for ZR 75-1 cells. Furthermore, although the most sensitive line, T-47D, had the highest level of PR, sensitivity to MPA was not correlated with PR levels among the responsive cell lines. More detailed studies were undertaken with the T-47D cell line. The growth-inhibitory response was confined to the progestins: MPA, ORG 2058, R5020, and progesterone, while androgens, estrogens, and glucocorticoids were without effect over the same concentration range (0.1-100 nM). MPA-induced growth inhibition was associated with a significant decrease in the proportion of S-phase cells with an accumulation of cells in the G0-G1 phase of the cell cycle. Cells began to accumulate in G0-G1 after 12 h of drug treatment and the effect was maximal by 24 h, i.e., maximal effects were observed during the first cell cycle following drug treatment. By contrast, significant accumulation in G0-G1 required exposure of MCF-7 cells to MPA for at least two cell cycle times, i.e., 48 h and the effect was still increasing at 96 h. Stathmokinetic studies revealed that in both cell lines accumulation in the G0-G1 phase was due to an MPA-induced increase in the G1 transit time. These data indicate that MPA and other progestins have direct growth inhibitory effects on estrogen receptor-positive and progesterone receptor-positive human breast cancer cells in vitro and these effects can be accounted for by a decrease in the rate at which cells traverse the G1 phase of the cell cycle.

Breast Neoplasms↗
PubMed1988

Ribonucleotide reductase M1 subunit in cellular proliferation, quiescence, and differentiation.

Ribonucleotide reductase catalyzes the first unique, rate-limiting step in DNA synthesis; both its large (M1) and small (M2) subunits are necessary for activity. While direct studies of M2 expression have previously shown a tight correlation with S phase, the kinetic features of M1-expressing cells have remained ill defined. Therefore we have, using immunofluorescence flow cytometry, analyzed changes in whole cell M1 levels and DNA content during various cell cycle and differentiation events. In asynchronous cultures M1 levels are sustained throughout the cell cycle, including G1 phase when M2 levels and ribonucleotide reductase catalytic activity are known to be very low. In contrast M1 is virtually absent from quiescent lymphocytes but is expressed following mitogen stimulation, shortly before S phase cells appear. M1 declines to low levels in "plateau phase" cultures, the major reduction occurring in cells with 2n (G0/G1) DNA content. HL-60 promyelocytic leukemia cells, induced into either myeloid or monocyte-macrophage differentiation, show a similar marked decrease in M1 levels concomitant with the cessation of cell division. We conclude that the M1 subunit of ribonucleotide reductase is constitutively expressed by cycling cells. It is acquired during stimulated transition from G0 to G1 and is lost during exit to G0 or terminal differentiation. This pattern of expression suggests that determination of cellular M1 content may be useful in distinguishing proliferating (including G1) and quiescent (including G0) cells in vivo.

Cell Differentiation↗
PubMed1987

Immunofluorescent quantification of ribonucleotide reductase M1 subunit and correlation with DNA content by flow cytometry.

The cytoplasmic enzyme ribonucleotide reductase is essential for DNA synthesis, and its activity is strongly correlated with cellular proliferation. This paper describes a flow cytometric technique for the simultaneous measurement of DNA content and the M1 subunit of ribonucleotide reductase. Data are presented for cycling cultured human leukemic lymphoblasts in which M1 is constitutively expressed, and peripheral blood lymphocytes in which it is only detectable with certainty after mitogen stimulation. The choice of fixation procedure strongly influenced the amount of M1 subunit detected. Paraformaldehyde (PF) at concentrations of 2% (w/v in PBS) or greater provided optimal results. Fixation at 37 degrees C was significantly more effective in preserving M1 than fixation at room temperature or 4 degrees C. These variables are shown to have affected cytoplasmic retention during postfixation processing. Their relevance to the flow cytometric measurement of other intracellular components by this procedure are discussed.

Cell Cycle↗
PubMed1986

Transferrin receptor cycling by human lymphoid cells: lack of effect from inhibition of microtubule assembly.

Flow cytometry was used to follow the uptake and release of fluorescein-conjugated ferro-transferrin by CCRF-CEM human T - leukaemia cells on an individual cell basis. Pretreatment with the stathmokinetic agent vincristine for 5 hr had no effects on the cycling of transferrin, although microscopic examination of the cells showed a substantial proportion to be arrested in metaphase with cytoplasmic dispersal of tubulin, indicating inhibition of microtubule assembly. Tubulin is therefore unlikely to play a major role in the transferrin cycle of exponentially-growing cells, despite earlier reports that the cytoskeleton is involved in receptor clustering and down regulation.

Cell Cycle↗