310
Biochimica et Biophysica Acta, 588 (1979) 310--321
© Elsevier/North-Holland Biomedical Press
BBA 29112
STUDIES ON THE MECHANISM O F ACTION O F H E X A M E T H Y L E N E BISACETAMIDE, A POTENT I N D U C E R O F E R Y T H R O L E U K E M I C DIFFERENTIATION
ROBERTA C. REUBEN Department of Human Genetics and Development and the Cancer Center Columbia University, New York, N Y 10032 (U.S.A.)
(Received April 2nd, 1979) Key words: Hexamethylene bisacetamide; Leukemia; Differentiation induction; (Friend cell)
Summary Hexamethylene bisacetamide (diacetyldiamino hexane) is a potent inducer of erythroid differentiation in murine erythroleukemia cells. Hexamethylene bisacetamide and the closely related pentamethylene bisacetamide were synthesized with radioactive labels in various portions of the molecule and the uptake, metabolism, and intracellular distribution determined. Bisacetamides are taken up b y the cell; an intracellular concentration equal to the extracellular concentration is achieved b y 6--8 h. C o m m i t m e n t to differentiation is n o t detected until at least 10 h after equilibration. Both uptake and c o m m i t m e n t to differentiate are concentration and temperature dependent. The majority of the c o m p o u n d is deacetylated upon cell entry and the acetate portion incorporated nonspecifically into lipid and protein. Acetate competes with the incorporation of hexamethylene bisacetamide into protein and lipid, but does not affect inducing activity. The diamine portion of the molecule is detected only in the cytoplasm, in a trichloroacetic acid-soluble and acetylated form, whereas the acetate moiety is detected in b o t h cytoplasm and nucleus and in both a trichloroacetic acid-soluble and insoluble form. The cellular uptake of diamines and bisacetamides (acetylated diamines) are similar, b u t acetylation of the diamine greatly increases inducing activity.
Introduction The virus-transformed murine erythroleukemia cell line established b y Dr. C. Friend has been used extensively as a model system for studying el,ythroid difA b b r e v i a t i o n s : HMBA, h e x a m e t h y l e n e bisacetamide; PMBA, pentamethylene bisacetamide.
311
ferentiation [1,2]. The transition from erythroid precursor (proerythroblast) to an advanced erythroid stage (orthochromic erythroblast) can be induced by inclusion of Me2SO in the culture medium [3]. Morphological differentiation is accompanied by the coordinate expression of erythroid functions such as synthesis and accumulation of globin mRNA [ 4 - 7 ] and globin polypeptides [8--10], induction of enzymes required for heme synthesis [11,12], and accumulation of hemoglobin [3] and the erythroid membrane protein, spectrin [13,14]. Attempts to understand the mechanism of action of Me2SO in inducing this alteration in cellular expression led to the discovery of numerous chemical compounds that share this activity [ 15,16]. Most compounds exhibiting inducing activity are small molecular mass compounds {less than 250 daltons), and contain both hydrophilic and hydrophobic planar portions of the molecule [15--18]. Additional inducers of erythroid differentiation that do not share these properties have been reported, such as ouabain [19], hemin [20], purine derivatives [21], short-chain fatty acids [18,22,23], and actinomycin D [24]. While many chemical compounds share the ability to induce erythroid differentiation in murine erythroleukemia cells there is considerable variation in the concentration required to achieve optimal activity and in the proportion of cells which differentiate upon exposure to the inducing agent. The exploration of molecular structures which might optimize inducing activity led to the discovery of a series of compounds, the bisacetamides or acetylated diamines, which are potent inducers of erythroid differentiation in murine erythroleukemia cells [17]. One of these, hexamethylene bisacetamide (HMBA), has been studied most extensively, since it induces virtually all cells of a sensitive strain to differentiate at a concentration 50-fold less than the effective concentration of Me2SO [17,25]. In addition, hexamethylene bisacetamide has been demonstrated to induce differentiation or to alter the phenotype in other cell lines, including neuroblastoma [26], a mesenchymal cell line derived from a glioblastoma multiforme [27], teratocarcinoma [28], L8 myoblasts (Miranda, A. and Reuben, R.C., unpublished data), and MDCK kidney epithelial cells [29]. Attempts to understand the mechanism of action of the bisacetamides in altering cellular expression have followed two routes. Alterations in inducing activity associated with alterations in molecular structure and configuration of the inducing compounds have been reported [18]. The interaction of the bisacetamides with murine erythroleukemia cells in terms of uptake, intracellular accumulation and metabolism, and the intracellular distribution of these compounds and their metabolic products is reported here. Materials and Methods Materials. The following radioactive compounds were purchased from New England Nuclear: [3H]acetic anhydride (NET-018H, 100Ci/mol); [1-14C]acetic anhydride (NEC-001A, 10 Ci/mol); [1,5-14C]cadaverine dihydrochloride (NEC410, 4.8 Ci/mol); [1,4-14C]putrescine dihydrochloride (NEC-150, 40 Ci/mol). Non-radioactive compounds were purchased from Aldrich Chemicals. Radioactively labelled bisacetamides were prepared by acetylation of the appropriate diamine [17].
312
Cell line and culture methods. Murine erythroleukemia cell line 745A was kindly provided b y Dr. C. Friend (The Mount Sinai School of Medicine, New York, NY). Clone 19 [30] derived from 745A was employed in these experiments. Cell culture and determination of hemoglobin-containing (benzidinereactive) cells was as previously reported [17,30]. Commitment to differentiation was assayed as described [25]. Uptake o f inducer. Radioactively labelled inducer was added to the culture medium at a final concentration of 5 mM. C o m p o u n d labelled with 3H was employed at 5 pCi/ml (1 Ci/mol). Compounds containing 14C were employed at 0.5 pCi/ml (100 mCi/mol). When both 3H and 14C inducing agents were employed in the same culture, the t w o c o m p o u n d s were mixed in equal proportions (2.5 mM each, 2.5 pCi/ml of 3H, and 0.25 pCi/ml of 14C). At appropriate times after initiation of culture, duplicate samples containing approximately 5 . 1 0 6 cells were transferred to 0°C, recovered by centrifugation 10 min at 800 × g at 4°C, and washed three times with cold phosphate-buffered saline. The cell pellet was drained well and stored at --20 ° C. In initial experiments, in which cells were not subjected to extensive washing and in which contamination with extracellular fluid was measured b y addition of [14C]inulin to the sample immediately prior to recovery of cells b y centrifugation, essentially identical results were obtained, indicating that bisacetamides are not detectably lost from the cell during washing of the cells at 0--4°C. Elimination of the necessity for employing [~4C]inulin to measure extracellular contamination made it possible to use doubly labelled compounds. Frozen cell pellets were resuspended in 0.22 ml of distilled H20 and frozen and thawed twice. Protein concentration was determined b y the method of L o w r y et al. [31]. For each time point one sample (0.2 ml aliquot) was solubilized in 1 ml of NCS (Amersham/Searle), incubated 18 h at 55°C, and radioactivity determined. To the duplicate sample, 0.2 ml was made 10% in trichloroacetic acid and trichloroacetic acid-insoluble material recovered b y centrifugation at 10 000 × g for 10 min at 4°C. The supernatant was removed and trichloroacetic acid-soluble radioactivity determined. The trichloroacetic acid-insoluble pellet was washed once with 5% trichloroacetic acid and solubilized in NCS for 18 h at 55°C and radioactivity determined. Radioactivity was normalized to cell protein. Cell fractionation. Nuclear and cytoplasmic fractions were separated by detergent solubilization as described [10]. When detergent solubilization of plasma membrane was to be avoided, cells were disrupted by nitrogen cavitation [32,33]. Solubility in organic solvents was determined using either 9 vols. of acetone (4 ° C) or equal volumes of chloroform/methanol (2:1). Results
Radioactively labelled bisacetamides In order to investigate the interaction of the bisacetamides with murine erythroleukemia cells the penta- and hexamethylene bisacetamides were synthesized with a radioactive label in different portions of the molecule. O O II II 3H3C--14C--NH--14CH2--(CH)n--14CH2--NH--14C--C3H3
313
Initial studies employed hexamethylene bisacetamide (HMBA) with 3H in the terminal methyl group of the acetate moiety. To investigate the fate of the other portions of the molecule, pentamethylene bisacetamide (PMBA) was synthesized with 3H in the methyl of acetate and 14C in the carbonyl of acetate or 14C in the internal polymethylene chain. Pentamethylene bisacetamide was employed for double-label experiments since 1,5-diaminopentane and not 1,6diaminohexane is commercially available with radioactively labelled methylene groups. PMBA is an equally potent inducer and there was no difference in results obtained with either compound.
Uptake of hexamethylene bisacetamide and commitment to differentiate The kinetics of uptake of HMBA was determined employing a compound radioactively labelled with 3H in the terminal methyl group of acetate. When cells are cultured in the presence of optimal concentrations of HMBA (5 raM), the compound enters the cell at a linear rate for 10--12 h after initiation of culture. Intracellular accumulation plateaus by 24--30 h, and cell-associated radioactivity, normalized to cell protein, remains unchanged for 1--5 days (Fig. la). When cells are transferred to inducer-flee medium at 2 h, the cell-associated HMBA-derived radioactivity decreases by approximately 25% during the first 2 h (Fig. la). The decrease in radioactivity per cell observed after this initial drop can be accounted for by an increase in cell number. Subsequent experiments revealed that the remaining fraction represented HMBA~lerived radioactivity in
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Fig. I . H M B A u p t a k e and c o m m i t m e n t t o d i f f e r e n t i a t i o n . Cells w e r e c u l t u r e d in t h e p r e s e n c e o f 5 m M H M B A l a b e l l e d w i t h 3 H in t h e t e r m i n a l m e t h y l g r o u p (5 ~ C i / m l ) . (a) H M B A u p t a k e . T o t a l intracellular a c c u m u l a t i o n w a s d e t e r m i n e d b y s o l u b i l i z a t i o n in N C S o f d u p l i c a t e cell s a m p l e s w i t h d r a w n at t h e indic a t e d t i m e s . P r o t e i n and r a d i o a c t i v e c o n t e n t w e r e d e t e r m i n e d f o r e a c h s a m p l e . T h e average values o f d u p l i c a t e s a m p l e s have b e e n p l o t t e d as a f u n c t i o n o f t i m e ( e e). 24 h after initiation of culture a p o r t i o n o f the cells w a s p l a c e d in fresh, p r e w a r m e d m e d i u m w i t h o u t H M B A p r e s e n t , a n d Intracellular H M B A - d e r i v e d r a d i o a c t i v i t y d e t e r m i n e d at t h e i n d i c a t e d t i m e s ( o . . . . . . o ) . (b) C o m m i t m e n t t o d i f f e r e n t i a t i o n . Cells c u l t u r e d in the p r e s e n c e o f 5 m M H M B A f o r v a r y i n g p e r i o d s o f t i m e w e r e transferred t o fresh m e d i u m w i t h o u t i n d u c e r . A f t e r a t o t a l o f 5 d a y s f r o m the t i m e o f i n i t i a t i o n o f cell c u l t u r e , t h e p r o p o r t i o n o f cells t h a t w e r e b e n z i d i n e r e a c t i v e ( c o n t a i n h e m o g l o b i n ) w a s d e t e r m i n e d and h a s b e e n p l o t t e d as a f u n c t i o n o f t i m e o f e x p o s u r e t o H M B A .
314
a trichloroacetic acid-insoluble form. If cells are cultured with or without 5 mM non-radioactive HMBA and the medium replaced at either 24 or 48 h with [methyl-3H]HMBA, the kinetics of uptake are identical whether or not the cells have been previously exposed to HMBA (data not shown). The temporal relationship of HMBA uptake and c o m m i t m e n t to differentiate was investigated. When murine erythroleukemia cells are cultured in the presence of inducer for varying periods of time before transfer to medium without inducer, a portion o f the culture is ' c o m m i t t e d ' , in that some cells continue the developmental process in the absence of inducer [25,35]. The percentage of cells which are benzidine reactive (contain hemoglobin) after a total of 5 days in culture increases with increased time of exposure to inducer. Commitment to differentiate is first detected at about 24 h, the time at which the intracellular accumulation of HMBA has achieved a maximal level (Fig. lb). Uptake, intracellular accumulation, and c o m m i t m e n t to differentiate are dependent upon extracellular concentration of inducer and upon temperature of incubation. At suboptimal concentrations (2 mM) both the rate of entry into the cell and the final concentration of cell-associated HMBA~lerived radio-
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Fig. 2. H M B A uptake and c o m m i t m e n t t o differentiate under suboptimal conditions o f concentration and t e m p e r a t u r e . Cells w e r e c u l t u r e d at 3 7 ° C in t h e p r e s e n c e o f 2 m M or 5 m M [methyl-SH]HMBA, o r a t 5°C w i t h 5 m M H M B A . T o t a l int~acellulax (a) a n d t r l e h l o r o a c e f l c acid-insoluble (b) H M B A - d e r i v e d r a d i o a c t i v i t y , a n d c o m m i t m e n t t o d i f f e r e n t i a t e (c), was d e t e r m i n e d f o r e a c h c u l t u z e at t h e i n d i c a t e d t i m e s . • -', 5 m M a n d 3 7 ° C , ~ . . . . . -~, 2 m M a n d 3 7 ° C , a n d X . . . . . . X, 5 m M a n d 5°C.
315 activity are reduced as compared to those observed at 5 mM. Cellular uptake is also diminished by culture at 5°C as compared to that observed at 37°C (Fig. 2a). A majority of the radioactivity from the methyl group of HMBA is converted to a form t h a t is insoluble in cold trichloroacetic acid (Fig. 2b). This conversion to trichloroacetic acid insolubility is reduced in rate and extent at suboptimal concentrations, although the proportion of material that becomes trichloroacetic acid insoluble is the same at both 2 mM and 5 mM. Conversion of HMBA-derived radioactivity to a trichloroacetic acid-insoluble form does not occur at 5 ° C. C o m m i t m e n t to differentiate was determined under the three conditions at which uptake was determined, namely 2 mM or 5 mM HMBA at 37°C, and 5 mM HMBA at 5°C {Fig. 2c). C o m m i t m e n t occurred at both concentrations when cultured at 37°C, although this parameter was also reduced in rate and extent at the lower concentration. C o m m i t m e n t did not occur when cultured at 5°C for periods up to 96 h, but the cells remained viable and retained the capacity to differentiate for up to 96 h when transferred to 37°C in the presence of inducer. Cellular uptake studies are performed on cell populations, whereas assays of c o m m i t m e n t and differentiation evaluate single cells. The proportion of cells induced to differentiate is reduced under suboptimal conditions, but those cells that become c o m m i t t e d appear to attain the same degree of morphological maturation and hemoglobin content as under optimal conditions. It is possible that there is a subpopulation of cells which are more sensitive to the action of inducer or accumulate it more readily. To determine if there is a permanent subpopulation of cells more sensitive to the inducing effects of HMBA, cells were cultured in the presence of 0, 2 mM, or 5 mM HMBA for 4 days to allow c o m m i t m e n t to differentiation to achieve maximal levels for each concentration tested. Cells were then cultured for 3 days in the absence of inducer so that c o m m i t t e d cells would have sufficient time to terminally differentiate. Surviving cells from each culture were then divided and exposed again to either 0, 2 mM, or 5 mM HMBA. The proportion of cells induced to differentiate was the same as upon initial exposure to inducer. Thus cells resisting the differentiation-inducing effect of 2 mM HMBA on first exposure were n o t noticeably more resistant to HMBA, in terms of proportion of cells differentiating, on second exposure {data not shown). Since uptake and c o m m i t m e n t are proportional to extracellular concentration, both processes might occur more rapidly if the extracellular concentration were increased for periods of time which are not cytotoxic. However, attempts to shorten the p r e c o m m i t m e n t phase by exposure to high concentrations of HMBA (5--50 mM) for short periods of time (24 h and less) did n o t result in a more rapid c o m m i t m e n t .
Analysis of different portions of the inducer To investigate the fate of the other portions of the molecule, pentamethylene bisacetamide was employed as inducer with 3H in the terminal m e t h y l of acetate and '4C in either carbonyl of acetate or in the internal p o l y m e t h y l e n e chain. These three portions of the molecule accumulate in the acid-soluble fraction with similar kinetics and equilibrate at concentrations equivalent to
316
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Biochimica et Biophysica Acta, 588 (1979) 310--321
© Elsevier/North-Holland Biomedical Press
BBA 29112
STUDIES ON THE MECHANISM O F ACTION O F H E X A M E T H Y L E N E BISACETAMIDE, A POTENT I N D U C E R O F E R Y T H R O L E U K E M I C DIFFERENTIATION
ROBERTA C. REUBEN Department of Human Genetics and Development and the Cancer Center Columbia University, New York, N Y 10032 (U.S.A.)
(Received April 2nd, 1979) Key words: Hexamethylene bisacetamide; Leukemia; Differentiation induction; (Friend cell)
Summary Hexamethylene bisacetamide (diacetyldiamino hexane) is a potent inducer of erythroid differentiation in murine erythroleukemia cells. Hexamethylene bisacetamide and the closely related pentamethylene bisacetamide were synthesized with radioactive labels in various portions of the molecule and the uptake, metabolism, and intracellular distribution determined. Bisacetamides are taken up b y the cell; an intracellular concentration equal to the extracellular concentration is achieved b y 6--8 h. C o m m i t m e n t to differentiation is n o t detected until at least 10 h after equilibration. Both uptake and c o m m i t m e n t to differentiate are concentration and temperature dependent. The majority of the c o m p o u n d is deacetylated upon cell entry and the acetate portion incorporated nonspecifically into lipid and protein. Acetate competes with the incorporation of hexamethylene bisacetamide into protein and lipid, but does not affect inducing activity. The diamine portion of the molecule is detected only in the cytoplasm, in a trichloroacetic acid-soluble and acetylated form, whereas the acetate moiety is detected in b o t h cytoplasm and nucleus and in both a trichloroacetic acid-soluble and insoluble form. The cellular uptake of diamines and bisacetamides (acetylated diamines) are similar, b u t acetylation of the diamine greatly increases inducing activity.
Introduction The virus-transformed murine erythroleukemia cell line established b y Dr. C. Friend has been used extensively as a model system for studying el,ythroid difA b b r e v i a t i o n s : HMBA, h e x a m e t h y l e n e bisacetamide; PMBA, pentamethylene bisacetamide.
311
ferentiation [1,2]. The transition from erythroid precursor (proerythroblast) to an advanced erythroid stage (orthochromic erythroblast) can be induced by inclusion of Me2SO in the culture medium [3]. Morphological differentiation is accompanied by the coordinate expression of erythroid functions such as synthesis and accumulation of globin mRNA [ 4 - 7 ] and globin polypeptides [8--10], induction of enzymes required for heme synthesis [11,12], and accumulation of hemoglobin [3] and the erythroid membrane protein, spectrin [13,14]. Attempts to understand the mechanism of action of Me2SO in inducing this alteration in cellular expression led to the discovery of numerous chemical compounds that share this activity [ 15,16]. Most compounds exhibiting inducing activity are small molecular mass compounds {less than 250 daltons), and contain both hydrophilic and hydrophobic planar portions of the molecule [15--18]. Additional inducers of erythroid differentiation that do not share these properties have been reported, such as ouabain [19], hemin [20], purine derivatives [21], short-chain fatty acids [18,22,23], and actinomycin D [24]. While many chemical compounds share the ability to induce erythroid differentiation in murine erythroleukemia cells there is considerable variation in the concentration required to achieve optimal activity and in the proportion of cells which differentiate upon exposure to the inducing agent. The exploration of molecular structures which might optimize inducing activity led to the discovery of a series of compounds, the bisacetamides or acetylated diamines, which are potent inducers of erythroid differentiation in murine erythroleukemia cells [17]. One of these, hexamethylene bisacetamide (HMBA), has been studied most extensively, since it induces virtually all cells of a sensitive strain to differentiate at a concentration 50-fold less than the effective concentration of Me2SO [17,25]. In addition, hexamethylene bisacetamide has been demonstrated to induce differentiation or to alter the phenotype in other cell lines, including neuroblastoma [26], a mesenchymal cell line derived from a glioblastoma multiforme [27], teratocarcinoma [28], L8 myoblasts (Miranda, A. and Reuben, R.C., unpublished data), and MDCK kidney epithelial cells [29]. Attempts to understand the mechanism of action of the bisacetamides in altering cellular expression have followed two routes. Alterations in inducing activity associated with alterations in molecular structure and configuration of the inducing compounds have been reported [18]. The interaction of the bisacetamides with murine erythroleukemia cells in terms of uptake, intracellular accumulation and metabolism, and the intracellular distribution of these compounds and their metabolic products is reported here. Materials and Methods Materials. The following radioactive compounds were purchased from New England Nuclear: [3H]acetic anhydride (NET-018H, 100Ci/mol); [1-14C]acetic anhydride (NEC-001A, 10 Ci/mol); [1,5-14C]cadaverine dihydrochloride (NEC410, 4.8 Ci/mol); [1,4-14C]putrescine dihydrochloride (NEC-150, 40 Ci/mol). Non-radioactive compounds were purchased from Aldrich Chemicals. Radioactively labelled bisacetamides were prepared by acetylation of the appropriate diamine [17].
312
Cell line and culture methods. Murine erythroleukemia cell line 745A was kindly provided b y Dr. C. Friend (The Mount Sinai School of Medicine, New York, NY). Clone 19 [30] derived from 745A was employed in these experiments. Cell culture and determination of hemoglobin-containing (benzidinereactive) cells was as previously reported [17,30]. Commitment to differentiation was assayed as described [25]. Uptake o f inducer. Radioactively labelled inducer was added to the culture medium at a final concentration of 5 mM. C o m p o u n d labelled with 3H was employed at 5 pCi/ml (1 Ci/mol). Compounds containing 14C were employed at 0.5 pCi/ml (100 mCi/mol). When both 3H and 14C inducing agents were employed in the same culture, the t w o c o m p o u n d s were mixed in equal proportions (2.5 mM each, 2.5 pCi/ml of 3H, and 0.25 pCi/ml of 14C). At appropriate times after initiation of culture, duplicate samples containing approximately 5 . 1 0 6 cells were transferred to 0°C, recovered by centrifugation 10 min at 800 × g at 4°C, and washed three times with cold phosphate-buffered saline. The cell pellet was drained well and stored at --20 ° C. In initial experiments, in which cells were not subjected to extensive washing and in which contamination with extracellular fluid was measured b y addition of [14C]inulin to the sample immediately prior to recovery of cells b y centrifugation, essentially identical results were obtained, indicating that bisacetamides are not detectably lost from the cell during washing of the cells at 0--4°C. Elimination of the necessity for employing [~4C]inulin to measure extracellular contamination made it possible to use doubly labelled compounds. Frozen cell pellets were resuspended in 0.22 ml of distilled H20 and frozen and thawed twice. Protein concentration was determined b y the method of L o w r y et al. [31]. For each time point one sample (0.2 ml aliquot) was solubilized in 1 ml of NCS (Amersham/Searle), incubated 18 h at 55°C, and radioactivity determined. To the duplicate sample, 0.2 ml was made 10% in trichloroacetic acid and trichloroacetic acid-insoluble material recovered b y centrifugation at 10 000 × g for 10 min at 4°C. The supernatant was removed and trichloroacetic acid-soluble radioactivity determined. The trichloroacetic acid-insoluble pellet was washed once with 5% trichloroacetic acid and solubilized in NCS for 18 h at 55°C and radioactivity determined. Radioactivity was normalized to cell protein. Cell fractionation. Nuclear and cytoplasmic fractions were separated by detergent solubilization as described [10]. When detergent solubilization of plasma membrane was to be avoided, cells were disrupted by nitrogen cavitation [32,33]. Solubility in organic solvents was determined using either 9 vols. of acetone (4 ° C) or equal volumes of chloroform/methanol (2:1). Results
Radioactively labelled bisacetamides In order to investigate the interaction of the bisacetamides with murine erythroleukemia cells the penta- and hexamethylene bisacetamides were synthesized with a radioactive label in different portions of the molecule. O O II II 3H3C--14C--NH--14CH2--(CH)n--14CH2--NH--14C--C3H3
313
Initial studies employed hexamethylene bisacetamide (HMBA) with 3H in the terminal methyl group of the acetate moiety. To investigate the fate of the other portions of the molecule, pentamethylene bisacetamide (PMBA) was synthesized with 3H in the methyl of acetate and 14C in the carbonyl of acetate or 14C in the internal polymethylene chain. Pentamethylene bisacetamide was employed for double-label experiments since 1,5-diaminopentane and not 1,6diaminohexane is commercially available with radioactively labelled methylene groups. PMBA is an equally potent inducer and there was no difference in results obtained with either compound.
Uptake of hexamethylene bisacetamide and commitment to differentiate The kinetics of uptake of HMBA was determined employing a compound radioactively labelled with 3H in the terminal methyl group of acetate. When cells are cultured in the presence of optimal concentrations of HMBA (5 raM), the compound enters the cell at a linear rate for 10--12 h after initiation of culture. Intracellular accumulation plateaus by 24--30 h, and cell-associated radioactivity, normalized to cell protein, remains unchanged for 1--5 days (Fig. la). When cells are transferred to inducer-flee medium at 2 h, the cell-associated HMBA-derived radioactivity decreases by approximately 25% during the first 2 h (Fig. la). The decrease in radioactivity per cell observed after this initial drop can be accounted for by an increase in cell number. Subsequent experiments revealed that the remaining fraction represented HMBA~lerived radioactivity in
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Fig. I . H M B A u p t a k e and c o m m i t m e n t t o d i f f e r e n t i a t i o n . Cells w e r e c u l t u r e d in t h e p r e s e n c e o f 5 m M H M B A l a b e l l e d w i t h 3 H in t h e t e r m i n a l m e t h y l g r o u p (5 ~ C i / m l ) . (a) H M B A u p t a k e . T o t a l intracellular a c c u m u l a t i o n w a s d e t e r m i n e d b y s o l u b i l i z a t i o n in N C S o f d u p l i c a t e cell s a m p l e s w i t h d r a w n at t h e indic a t e d t i m e s . P r o t e i n and r a d i o a c t i v e c o n t e n t w e r e d e t e r m i n e d f o r e a c h s a m p l e . T h e average values o f d u p l i c a t e s a m p l e s have b e e n p l o t t e d as a f u n c t i o n o f t i m e ( e e). 24 h after initiation of culture a p o r t i o n o f the cells w a s p l a c e d in fresh, p r e w a r m e d m e d i u m w i t h o u t H M B A p r e s e n t , a n d Intracellular H M B A - d e r i v e d r a d i o a c t i v i t y d e t e r m i n e d at t h e i n d i c a t e d t i m e s ( o . . . . . . o ) . (b) C o m m i t m e n t t o d i f f e r e n t i a t i o n . Cells c u l t u r e d in the p r e s e n c e o f 5 m M H M B A f o r v a r y i n g p e r i o d s o f t i m e w e r e transferred t o fresh m e d i u m w i t h o u t i n d u c e r . A f t e r a t o t a l o f 5 d a y s f r o m the t i m e o f i n i t i a t i o n o f cell c u l t u r e , t h e p r o p o r t i o n o f cells t h a t w e r e b e n z i d i n e r e a c t i v e ( c o n t a i n h e m o g l o b i n ) w a s d e t e r m i n e d and h a s b e e n p l o t t e d as a f u n c t i o n o f t i m e o f e x p o s u r e t o H M B A .
314
a trichloroacetic acid-insoluble form. If cells are cultured with or without 5 mM non-radioactive HMBA and the medium replaced at either 24 or 48 h with [methyl-3H]HMBA, the kinetics of uptake are identical whether or not the cells have been previously exposed to HMBA (data not shown). The temporal relationship of HMBA uptake and c o m m i t m e n t to differentiate was investigated. When murine erythroleukemia cells are cultured in the presence of inducer for varying periods of time before transfer to medium without inducer, a portion o f the culture is ' c o m m i t t e d ' , in that some cells continue the developmental process in the absence of inducer [25,35]. The percentage of cells which are benzidine reactive (contain hemoglobin) after a total of 5 days in culture increases with increased time of exposure to inducer. Commitment to differentiate is first detected at about 24 h, the time at which the intracellular accumulation of HMBA has achieved a maximal level (Fig. lb). Uptake, intracellular accumulation, and c o m m i t m e n t to differentiate are dependent upon extracellular concentration of inducer and upon temperature of incubation. At suboptimal concentrations (2 mM) both the rate of entry into the cell and the final concentration of cell-associated HMBA~lerived radio-
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Fig. 2. H M B A uptake and c o m m i t m e n t t o differentiate under suboptimal conditions o f concentration and t e m p e r a t u r e . Cells w e r e c u l t u r e d at 3 7 ° C in t h e p r e s e n c e o f 2 m M or 5 m M [methyl-SH]HMBA, o r a t 5°C w i t h 5 m M H M B A . T o t a l int~acellulax (a) a n d t r l e h l o r o a c e f l c acid-insoluble (b) H M B A - d e r i v e d r a d i o a c t i v i t y , a n d c o m m i t m e n t t o d i f f e r e n t i a t e (c), was d e t e r m i n e d f o r e a c h c u l t u z e at t h e i n d i c a t e d t i m e s . • -', 5 m M a n d 3 7 ° C , ~ . . . . . -~, 2 m M a n d 3 7 ° C , a n d X . . . . . . X, 5 m M a n d 5°C.
315 activity are reduced as compared to those observed at 5 mM. Cellular uptake is also diminished by culture at 5°C as compared to that observed at 37°C (Fig. 2a). A majority of the radioactivity from the methyl group of HMBA is converted to a form t h a t is insoluble in cold trichloroacetic acid (Fig. 2b). This conversion to trichloroacetic acid insolubility is reduced in rate and extent at suboptimal concentrations, although the proportion of material that becomes trichloroacetic acid insoluble is the same at both 2 mM and 5 mM. Conversion of HMBA-derived radioactivity to a trichloroacetic acid-insoluble form does not occur at 5 ° C. C o m m i t m e n t to differentiate was determined under the three conditions at which uptake was determined, namely 2 mM or 5 mM HMBA at 37°C, and 5 mM HMBA at 5°C {Fig. 2c). C o m m i t m e n t occurred at both concentrations when cultured at 37°C, although this parameter was also reduced in rate and extent at the lower concentration. C o m m i t m e n t did not occur when cultured at 5°C for periods up to 96 h, but the cells remained viable and retained the capacity to differentiate for up to 96 h when transferred to 37°C in the presence of inducer. Cellular uptake studies are performed on cell populations, whereas assays of c o m m i t m e n t and differentiation evaluate single cells. The proportion of cells induced to differentiate is reduced under suboptimal conditions, but those cells that become c o m m i t t e d appear to attain the same degree of morphological maturation and hemoglobin content as under optimal conditions. It is possible that there is a subpopulation of cells which are more sensitive to the action of inducer or accumulate it more readily. To determine if there is a permanent subpopulation of cells more sensitive to the inducing effects of HMBA, cells were cultured in the presence of 0, 2 mM, or 5 mM HMBA for 4 days to allow c o m m i t m e n t to differentiation to achieve maximal levels for each concentration tested. Cells were then cultured for 3 days in the absence of inducer so that c o m m i t t e d cells would have sufficient time to terminally differentiate. Surviving cells from each culture were then divided and exposed again to either 0, 2 mM, or 5 mM HMBA. The proportion of cells induced to differentiate was the same as upon initial exposure to inducer. Thus cells resisting the differentiation-inducing effect of 2 mM HMBA on first exposure were n o t noticeably more resistant to HMBA, in terms of proportion of cells differentiating, on second exposure {data not shown). Since uptake and c o m m i t m e n t are proportional to extracellular concentration, both processes might occur more rapidly if the extracellular concentration were increased for periods of time which are not cytotoxic. However, attempts to shorten the p r e c o m m i t m e n t phase by exposure to high concentrations of HMBA (5--50 mM) for short periods of time (24 h and less) did n o t result in a more rapid c o m m i t m e n t .
Analysis of different portions of the inducer To investigate the fate of the other portions of the molecule, pentamethylene bisacetamide was employed as inducer with 3H in the terminal m e t h y l of acetate and '4C in either carbonyl of acetate or in the internal p o l y m e t h y l e n e chain. These three portions of the molecule accumulate in the acid-soluble fraction with similar kinetics and equilibrate at concentrations equivalent to
316
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