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Activation of protein kinase C induces differentiation in the human T-lymphoblastic cell line MOLT-3.

We attempted to determine whether or not activation of calcium phospholipid-dependent protein kinase C (PKC) is associated with the induction of differentiation by 12-O-tetradecanoylphorbol-13-acetate (TPA) in the human T-lymphoblastic cell line MOLT-3. PKC activities were assayed in MOLT-3 and its five subclones resistant to TPA-induced cell differentiation. The cytosolic PKC activities of TPA-resistant subclones were 36-53% of that of the parental MOLT-3 cells. TPA treatment led to a rapid decrease in PKC activities in the cytosol, together with a concomitant increase in PKC activities in the particulate fraction, in both MOLT-3 and a TPA-resistant subclone. Thus, translocation of PKC from the cytosol to the membrane occurred following treatment with TPA, in both cell lines. However, the amount of PKC translocated from the cytosol to particulate fraction for 60 min in a TPA-resistant subclone was about 20% of that of the parental MOLT-3 cells. These findings suggest that the quantity of cytosolic PKC activity and the extent of translocation may relate to responses to TPA-induced cell differentiation in this T-cell line.

TPA, a most potent tumour promoter phorbol ester, exerts its effect on tissues and cultured cells (Diamond et al., 1980). The initial event in its action involves binding to the specific receptors on cell membrane, now identified to be calcium phospholipid-dependent PKC (Berridge, 1984;Niedel et at., 1983;Nishizuka et al., 1984;Parker et al., 1984;Sando & Young, 1983;Shoyab & Todaro, 1980). We reported TPA-induced differentiation in human malignant T-cell lines MOLT-3 and Jurkat and analysed the processes in the differentiation, using T-cell differentiation markers such as cell proliferation, E rosette formation, terminal deoxynucleotidyl transferase activity, monoclonal OKT antigen expression and morphological changes (Nagasawa & Mak, 1980, 1982Nagasawa et al., 1981a, b).
Using MOLT-3 and its subclones resistant to TPA induction, we also found that the receptors for phorbol ester play an important role at the initial process of induction of cell differentiation (Mayumi et al., 1988). Our main interest has been whether activation of PKC mediates the signals for differentiation as it does in the process of stimulation or proliferation in various cell systems (Kaibuchi et al., 1983;Kajikawa et al., 1983;Malaisse et al., 1983;Rozengurt et al., 1984), including human mature T-lymphocytes (Isakov et al., 1987;Manger et al., 1987).
In the induction of differentiation in the promyelocytic leukaemia cell line HL-60, a line most often used as a model of differentiation, some investigators suggested that TPA exerts an induction effect through PKC as a signal mediator (Anderson et al., 1985;Vandenbark et al., 1984), whereas others refuted this (Kreutter et al., 1985). Vandenbark et al. (1984) first proposed that the TPA-induced maturation of HL-60 might be mediated by the activation of intracellular PKC whereas Kreutter et al. (1985) observed a dissociation of the activation of PKC and the induction of cell maturation, determined using I-oleoyl-2-acetylglycerol, a synthetic compound which also directly activates PKC. However, little is known of the role of PKC in the induction of T-lymphocyte differentiation.
Activation of PKC is associated with its translocation from the cytosol to the membrane (Kraft et al., 1982;Kraft & Anderson, 1983). Kraft et al. (1982) and Kraft and Anderson (1983) demonstrated that TPA caused a translocation of PKC from the cytosol to the membrane in intact cells and these data were supported by other investigators (Shoji et al., 1987). Homma et al. (1986) also found that a translocation of PKC from the cytosol to the membrane fractions occurred in HL-60 cells, in response to TPA, whereas it did not occur in the TPA-resistant HL-60 variant cells, hence the translocation of PKC was presumed to be Correspondence: Y. Yamauchi. closely related to the TPA-induced differentiation in HL-60.
We have now examined the role of PKC, in particular its activation and subcellular distribution, in the TPA-induced differentiation of MOLT-3.

Cell culture
The human T-lymphoblastic cell line MOLT-3 was obtained from E. Gelfand (Hospital for Sick Children, Toronto, Canada). MOLT-3 subclones (ROI, R02, R03, R04 and R05) resistant to the growth inhibition effect by TPA were obtained from colonies formed in 0.8% methylcellulose containing 16nM TPA and 15% fetal calf serum (FCS), as described (Mayumi et al., 1988). These cells were maintained in RPMI 1640 medium supplemented with 10% FCS, 100mgl-1 streptomycin and 100Uml1penicillin. The concentration of acetone used in the cell culture did not exceed 0.01%.

Terminal deoxynucleotidyl transferase assay
The terminal deoxynucleotidyl transferase (TdT) activities in MOLT-3 cells and its subclones were measured by biochemical assay, as described (Okamura et al., 1978). Subcellular fractionation MOLT-3 or TPA-resistant cells (3-6 x 107 cells) maintained without TPA for at least a month were used in the following study. All subsequent steps were done at 4 C. The cells were washed twice with divalent cation-free PBS, resuspended in 3 ml homogenising buffer (20mM Tris, pH 7.5, 2mM EDTA, 0.5mM EGTA, 0.33 M sucrose, 2mM phenylmethylsulphonyl fluoride (PMSF) and 50 mM 2-mercaptoethanol) and sonicated with a Branson Model Sonifier for 45s at 20W.
The homogenates were centrifuged for 60min at 100,000g and the supernatant served as the cytosol fraction. The pellets were washed with homogenising buffer and recentrifuged for 60min at 100,000g. The pellet was used for the particulate fraction. PKC in the particulate fraction was extracted with 0.5% Nonidet P40, overnight on ice.
PKC from the cytosol fractions was partially punrfied by applying cell extracts from MOLT-3 and the TPA-resistant subclone ROI to a small DEAE-sepharose column (0.9 x 2.4 cm) equilibrated with buffer A (20mM Tris, pH 7.5, 2mM EDTA, 0.5mM EGTA and 2mM PMSF). After sample application, the column was washed with 30ml of buffer A and the enzyme was eluted with a linear gradient of NaCI (0-0.3M, total volume of 30ml, flow rate 60mlh-1). Fractions of 1.0ml were collected and S0pl of each fraction was used to measure the activity of PKC. On the basis of the elution profile obtained, the cytosol and particulate preparations were fractionated on a DEAE-sepharose column, using a one step with 0.15 M NaCl in 4ml of buffer A, and PKC activities were determined. Subcellular distribution of PKC Intact MOLT-3 cells and the TPA-resistant subclone ROI (3 x107 cells) were preincubated at 37 C for 30min, then TPA was added at a final concentration of 16nM. The cells were incubated for various time periods up to 60min and washed twice with ice-cold PBS. PKC in the cytosol and the particulate fraction was partially purified on a DEAEsepharose column, using a one step with 0.15 M NaCI, and PKC activity was determined.
PKC assay PKC activity was determined by measuring the incorporation of 32P from _y-32P-ATP into histone HI, as described (Kikkawa et al., 1982). The reaction mixture, in a final volume of 250 pl, consisted of 80pgml-1 PS, 20mM Tris (pH 7.5), 0.1 mM CaCl2, 5mM magnesium acetate, 2.5 nmol y-32P-ATP (100c.p.m.pmoli), 50pg of histone HI and 50y of sample in the absence or presence of I jgmmlP TPA. Incubation was carried out for 5 min at 30'C. The reaction was terminated by the addition of 25% trichloroacetic acid. The acid-precipitated matenrals were collected on a membrane filter and counted for radioactivity. PKC activity was determined by subtracting the activity measured in the absence of TPA from that measured in its presence and expressed as nmol Of 32P transferred to histone H1 per min at 30-C per 108 cells. Protein was estimated by the method of Bradford (1976). Bovine serum albumin was used as the standard.

Results
Characteristics of TPA-resistant subclones The proliferation of MOLT-3 in the presence of 16nM TPA was reduced with 3 days of culture, after which the cells grew slowly up to day 9, with a viability exceeding 90%, as determined by trypan blue dye exclusion. Thus, the TPA was not toxic to these MOLT-3 cells (Figure la). The five TPAresistant subclones of MOLT-3 obtained in methylcellulose containing TPA grew equally well in suspension cultures, with or without 16nM TPA (Figure lb). The form and size of these TPA-resistant subclones did not differ from those of the parental MOLT-3. The resistance of those clones to TPA was not lost for up to several months, even in continuous culture without TPA.
The presence of TdT is characteristic of prothymocytes and is absent in mature T-lymphocytes (Bollum, 1979). The level of the enzyme of MOLT-3 was reduced dramatically in the presence of 16nM TPA, reaching a level of 12% of the control culture at 3 days, whereas the level of the enzyme of TPA-resistant subclones remained high after TPA stimulation for 3 days (Table I). These results indicate that the parental MOLT-3 reaches a more differentiated state whereas the TPA-resistant subclones remain immature in the presence of TPA.  Elution profile of cvtosolic PKC of MOLT-3 and TPAresistant subclone ROJ Cytosol preparations of MOLT-3 were fractionated on DEAE-sepharose columns using a linear gradient and column fractions were assayed for PKC activity. The PKC activity eluted at a concentration of 0.06-0.16M with a peak at 0.10M NaCI (Figure 2a). Cytosol preparations of ROI showed an elution profile of PKC, in a similar fashion and with a peak at 0.10M NaCl (Figure 2b). However, the amount of PKC activity in ROI was 40% of that of the parental MOLT-3. The baseline of PKC activity in ROI was lower than that of MOLT-3, thereby indicating that PKC in ROI was not already activated. To determine whether PKC activities of TPA-resistant subclones were less than those of Fugre 2 PKC activities on DEAE-sepharose chromatography of MOLT-3 (a) and TPA-resistant subclone (ROI) (b). Cytosol from 108 Ocells (MOLT-3, 10.0mg; RO1, 10.1 mg) were applied to and eluted from the column, as described in Materials an wtods. PKC activity was determined as described with 50d1 aliquots from the indicated effluent in the presence (@-*) or absence (Q-Q) of Iugmrl-TPA, with 0.1mM Ca2+ and 80pgml-1 PS and expressed as c.p.m. 32P incorporated into histone HI for 5 min per 50.ul sample.

Effect of TPA on subcellular distribution of PKC
To investigate the activation of PKC, the effect of TPA on the subcellular distribution of PKC in MOLT-3 and the TPA-resistant subclone ROI was examined (Figure 3). Most (98-99%) of the PKC activity was found in the cytosol and there was little PKC activity in the particulate fraction, in both cell lines. The stimulation with 16 nM TPA in the parental MOLT-3 resulted in a 50% decrease in cytosolic PKC activity within 5min, followed by a gradual decline to 10% of the initial level at 60 min ( Figure 3a) and a concomitant increase in PKC activity in the particulate fraction occurred (Figure 3b). A similar change of subcellular PKC distribution also occurred in ROI. Thus, the PKC activities in the cytosol and particulate fraction changed inversely, in both cell lines and in a time dependent manner, indicating that the translocation of PKC from the cytosol to the particulate fraction was caused by TPA Fugwe 3 Time course of PKC activities in the cytosolic (a) and particulate (b) fraction. MOLT-3 (@-*) and ROI (0 O) were incubated for the indicated times at 37-C with 16nM TPA in 0.0005% DMSO. Each point is the mean+s.e. of three separate experiments. PKC activity is expressed as nmol 32P incorporated into histone HI per 108 cells.
stimulation. However, the amount of decrease for 60 mn was 0.57nmolmin-' per 108 cells in ROI, 24% of 2.40nmolmin-m per 108 cells in MOLT-3 ( Figure 3a) and the amount of increase for 60min was 0.51 nmolminper 108 cells in ROI, 20% of 2.60nmolmin-' per 108 cells in MOLT-3 (Figure 3b). There was no detectable PKC activity in the cytosol and particulate fractions of both MOLT-3 and RO1 at 24, 72 and 120h after addition of 16nM TPA (data not shown). DMSO (0.0005%) had no apparent effect on translocation in MOLT-3 or the TPA-resistant subclones (data not shown).
To investigate the role of phorbol ester receptors or PKC in case of TPA-induced differentiation, a comparison between TPA-sensitive and resistant subclones should be of great use.
The TPA-resistant subclones from MOLT-3 proliferated in either the presence or absence of TPA. The TPA-resistance was also confirmed by the assay of TdT activities. TdT activities in these subclones remained high even in the u presence of TPA, whereas they were reduced in the sensitive parental MOLT-3 cells in 3 days' culture with TPA. These results indicated that the TPA-resistant subclones were not induced to differentiate by TPA and remained immature. In previous work, we found that the amount of phorbol ester binding to TPA-resistant subclones from MOLT-3 was about half that of the parental MOLT-3, presumably due to a low concentration of receptors for phorbol esters, as assayed by Scatchard analysis (Mayumi et al., 1988). Thus, we concluded that the number of receptors for phorbol esters played an important role in the induction of differentiation in MOLT-3 cells by TPA. Several studies have shown that the receptors for phorbol esters are copurified with PKC (Ashendel et al., 1983;Niedel et al., 1983). The enzyme is now thought to be a major receptor for phorbol esters (Sando & Young, 1983;Sharkey et al., 1984) and also one of the major signal mediators from the cell membrane to the nucleus (Nishizuka, 1984;Berridge, 1984). This prompted us to investigate whether PKC was related to the TPAinduced differentiation in MOLT-3. The levels of PKC in the cytosol in five TPA-resistant subclones were about half those in the parental MOLT-3. A similar result is shown in Figure  2, where the baseline of PKC activity without TPA is also lower in the TPA-resistant subclone ROI than in the parental MOLT-3. These results may reflect a decrease in number of phorbol ester receptors in TPA-resistant subclones of MOLT-3, as already noted (Mayumi et al., 1988). Phorbol esters with tumour-promoting activity cause a translocation of PKC from the cytosol to the membrane fraction in various cell systems (Kraft et al., 1982;Kraft & Anderson, 1983;Shoji et al., 1987) including human mature T-ells (Isakov et al., 1987;Manger et al., 1987). In cell differentiation systems, however, the role of PKC is not well understood. In the human promyelocytic leukaemia cell line HL-60, Vandenbark et al. (1984) found that the TPAinduced differentiation was mediated by PKC, whereas other investigators demonstrated that mediator(s) other than or in addition to the activation of PKC may be required for the induction of differentiation (Kreutter et al., 1985). In contrast, however, the role of PKC in TPA-induced differentiation in T-lymphocytes is less well understood. We found that a translocation of PKC oceurred in both TPA-resistant cells and TPA-sensitive MOLT-3 cells as seen in other cells (Kraft et al., 1982;Shoji et al., 1987). However, it should be emphasise that the amount of PKC translocated from the cytosol to the particulate fraction for 60min in ROI was only 20% of that in the MOLT-3 cells. The amount of cytosolic PKC and of its translocation may be a main factor associated with the induction of cell differentiation. These results differ from those shown by Homma et al. (1986), in that the level of cytosolic PKC in TPA-resistant HL-60 variant cells was as high as that in TPA-sensitive HL-60 cells and that translocation of PKC to the membrane did not occur in the resistant cells. In contrast, the results obtained by Shoji et al. (1987), who used the acute myeloblastic leukaemia cell line KG-I, are close to our observations. They showed that cytosolic PKC activity in the TPA-resistant subclone KG-la was about one third of that in the parental TPA-sensitive KG-I cells, despite similar patterns of translocation of PKC, in both lines. These discrepancies may be related to different cell systems. With respect to the phorbol ester receptors, there is also a difference between MOLT-3 and HL-60. The down regulation of phorbol ester receptors by TPA was seen both in TPA-resistant cells and in the parental MOLT-3 (Mayumi et al., 1988), whereas it was not observed in TPA-resistant cells from HL-60 (Solanki et al., 1981). The down regulation of phorbol ester receptors was observed within 15min after stimulation with TPA (Mayumi et al., 1988). The precise relationship between the down regulation of phorbol ester binding and PKC translocation is unclear, but both events may be related.
The role of PKC after the onset of cell differentiation is also unclear. There was no detectable PKC activity in the cytosol and particulate fractions for up to 120h after treatment with 16nM TPA, in both MOLT-3 and ROI. These results may reflect the continued translocation of PKC from the cytosol to the membrane and the degradation of membrane-associated PKC (Ballester & Rosen, 1985).
While this study provides no direct evidence that PKC translocation from the cytosol to the membrane fraction causes differentiation in MOLT-3, nevertheless, the existence of differences in the amount of cytosolic PKC, the amount of PKC translocation and the number of phorbol ester receptors between TPA-resistant subclones and the parental MOLT-3 cells do suggest that PKC plays an important role in the induction of differentiation by TPA in this Tlymphoblastic cell line MOLT-3.