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Adsorption of dactinomycin to nitrocellulose filters
Preliminary notes Adsorption of dactinomycin to nitrocellulose filters S. NORDLING,
H. MIETTINEN
and L. SAXEN,
Third Department of Pathology and Department Zoology, University of Helsinki, Finland
of
Summary. Dactinomycin is strongly adsorbed to nitrocellulose filters and some of the adsorbed material is slowly released. This affects both the growth and development of embryonic explants and the nucleotide incorporation of HeLa cells cultivated on pretreated filters.
Nitrocellulose (Millipore) filters are widely used in experimental biology both for sterile filtration and for tissue culture work. We obtained varying results in experiments on the effect of dactinomycin treatment on embryonic tissues separated by these filters in vitro cultures. This suggestedthat dactinomycin was adsorbed to the filter, then slowly releasedand incorporated in the tissue. Hence experiments were designed to investigate this. Material and Methods In transfilter experiments embryonic metanephric mesenchvme and spinal cord were cemented on both sides of a “TA” Millipore filter and cultured for 72 h in Eagle’s Minimum Essential Medium (MEM) supplemented with 10 % fetal calf serum (FCS) [l, 2,4]. Viability and differentiation was evaluated by light microscopy of serially sectioned explants. Filters were treated with SH-dactinomvcin in MEM containing 10 11 FCS for 1 h at 37”C, and then rinsed 3 times. For autoradiography explants were fixed in Carnoy’s solution, dehydrated in ethanol and cleared with benzene, embedded in paraffine and sectioned. Kodak AR 10 stripping film was used. For the evaluation of adsorption and release of dactinomycin, 1 cm2 pieces of 25 pm thick nitrocellulose filters (type TA, Millipore, Bedford, Mass.) were placed in test tubes containing 1 ml 3H-dactinomycin (Actinomycin D (aH), spec. act. 3.7 Ci/mmol; Schwarz/ Mann. Oranaeburg. N.Y.) in MEM for 30 min. Filters were -then -rinsed 3 times with phosphatebuffered saline (PBS). The radioactivitv was determined by liquid scintillation spectrometry. Five ml of cultivation medium containing 10 % FCS and 0.04 nmole/ml SH-dactinomycin or serum-free medium with 0.04 nmoleiml 8H-dactinomvcin and 0.36 nmoIe/ml non-radioactive dactinomycin were sterilized by filtration through a 13 mm diameter, 150 ,urn thick nitrocellulose filter (Millipore, type GS) fitted in a plastic holder (Swinnex, Millipore).
469
Twenty 1 cm2 pieces of TA filter were placed in 0.4 ,&i/ml SH-dactinomycin in 20 ml of PBS, incubated for 30 min and rinsed as above. One filter was placed in each tube with 2 ml MEM and incubated at 37°C. After 0 to 7 days, filters were removed and rinsed in PBS. Nitrocellulose filters were treated with dactinomycin in PBS for 30 min, rinsed 3 times with PBS and placed in 35 mm Petri dishes (Falcon Plastics, Oxnard, Calif.). Trypsinized HeLa cells were seeded onto these filters and cultivated for 3 days in MEM supplemented with 10% newborn calf serum. 3Huridine (NEN) was added to give a final concentration of 1 &i/ml. After 1 h the filters were removed and rinsed. The radioactivity was measured in half of the filters and the morphology of the cells was evaluated on the other half of the filters.
Results
A 1 cm2 filter with a volume of only about 0.0025 cm3and weighing about 1 mg removed 10-22~~ of the dactinomycin from a volume of 1.0 ml. More dactinomycin was removed from a serum-free than from a serumcontaining medium. The concentration of dactinomycin was 100-200 times greater in the filter than in the medium (table 1). About 5% of the dactinomycin adsorbed onto nitrocellulose filters was released during the first hour of incubation and about. 30% within one day with little change during further incubation. When a dactinornycincontaining solution was sterilized by filtration through a nitrocellulose filter there was a decrease in concentration, especially if the medium was serum-free (fig. 1). HeLa cells cultivated on filters treated with Table 1. Adsorption of dactinom,ycin to nitrocellulose ,filters Percentage removed by filter Initial cont. in medium (nmole/ ml) 0.004 0.04 0.4 4
Serumcontaining medium 13
16 II
10
Serumfree medium 15 19 22 14
Cont. in filter (nmole/g j1S.E.) --__ SerumconSerumtaining free medium medium 0.49 +0.04 0.54 to. I1 6.2 kO.5 7.01 0.6 45&10 83 f 1 380 & 30 580 4 30 -Exptl Cell Rrs HZ (1973)
470
Preliminary notes
I
2
3
4
5
Fig. 1. Abscissa: effluent volume from syringe, ml; ordinate: dactinomycin left in each ml of effluent, %. H, 0.4 nmole/ml in MEM; 0, 0.04 nmole/ml containing 10 % FCS. Amount of dactinomycin left after sterile filtration with nitrocellulose filters.
adsorbed to the filter seems to correlate fairly well with the concentration of dactinomycin in the medium. With the concentrations used no saturation effect was obtained. The adsorption of dactinomycin to nitrocellulose filters is quantitatively similar to that of protamine sulphate [3], but the mechanism of binding of dactinomycin to the filter is not known. The growth of both monolayer cells and tissue explants on dactinomycin-treated filters is severely affected. Embryonic tissues have been previously cultivated on nitrocellulose filters in the presence of dactinomycin [5, 61 and apparently, after removal of the dactinomycin, the filter has not been changed. It is difficult to evaluate whether this had any effect on the results of these experiments. Our conclusion is that whenever dactinomycin is used in embryological experiments
0.04 nmole/ml dactinomycin incorporated less 3H-UdR (1 600 cpm) than controls (8 400 cpm), and the morphology of the cells was poor. This experiment was repeated 3 times with qualitatively similar results. When spinal cord and metanephric mesenthyme were cultivated in a transfilter position on a 3H-dactinomycin-treated nitrocellulose filter there was no tubule formation (8 explants) in contrast to the tubule positive controls (9 explants). Autoradiograms showed grains on nuclei of both the spinal cord and of the mesenchyme (fig. 2). In these experiments the filters released so much dactinomycin that the medium concentration was 10 nCi/ml or 0.0027 nmole/ml. Discussion
The results indicate that dactinomycin is adsorbed to Millipore filters and that filter pieces can reduce the concentration of dactinomycin. Some of the adsorbed dactinomycin is released from the filter. The amount Exptl Cell Res 82 (1973)
Fig. 2. Uptake of 3H-dactinomycin by explants located on SH-dactinomycin-treated filter. Kodak AR 10 stripping film, exposure time 2 weeks, x 1 000. Spinal cord is located below and mesenchyme above the filter.
Preliminary notes
nitrocellulose filters have to be changed, since otherwise the duration of a pulse treatment may be longer than intended or, if the treatment is continuous, the local dactinomycin concentration may exceedthe bulk one. Furthermore, if diluted dactinomycin solutions are sterilized by filtration through nitrocellulose filters the final concentration may decreaseconsiderably. Grants: The Sigrid Juselius Foundation Foundation for Cancer Research, Finland.
and the
47 I
[2]. It is possible that fewer cells arrested in G2 in the older cultures becausea substance(s) was depleted in the root that is normally supplied by other plant organs. In the early stages of seedling growth the cotyledons are the chief source of most cell nutrients and consequently, they are most suspect as the tissue origin of the postulated substance(s) affecting cell arrest in G2. The experiments described in this paper were designed to test if the cotyledons influence cell arrest in G2 in root meristems of Pisum.
References 1. Grobstein, C, Exptl cell res 10 (1956) 424. 2. Jainchill, J, Saxen, L & Vainio, T, J embryo1 exptl morph01 12 (1964) 597. 3. Nordling, S, Miettinen, H, Wartiovaara, J & Saxen, L, J embryo1 exutl morph01 26 (1971) 231. 4. Sax& L & Sakseia, E, Exptl cell res 66 (1971) 349. 5. Wessells, N K, Devel biol 9 (1964) 92. 6. Wessells, N K & Wilt, F H, J mol biol 13 (1965) 767. Received June 25, 1973 Revised version received August 20, 1973
Cell arrest in G2 in root meristems:
A control factor from the cotyledons L. S. EVANS and J. VAN’T HOF, Biology Department, Brookhaven National Laboratory, Upton, N. Y. 11973, USA Summary. A substance promoting cell cycle arrest in G2 in the root meristem is demonstrated. This substance is produced in the cotyledons and is transported to the root.
Arrest in the mitotic cycle by meristematic cells in cultured primary root tips after protracted carbohydrate starvation is a nonrandom process [l]. Few, if any, cells stop during DNA synthesis and none arrest during mitosis. The probability of arrest in Gl and in G2 is not always equal. For example, in freshly excised carbohydrate-deficient meristems of Vicia faba the proportion of cells arrested, G 1 : G2, was approx. 1: 3 while that of older cultured meristems was about 1: 1
Materials and Methods The general procedures used in our experiments have been described previously [3]. Seeds of Pisum satiaum were surface sterilized in undiluted Clorox bleach containing 5.2 X sodium hypochlorite for 5 min, washed with water, and placed in sterile vermiculite. Three and 7 days after planting, l-l.5 cm primary root tips were placed into culture medium [6] with or without sucrose. Roots from seedlings were fixed after treatment in ethanol/acetic acid (3 : I, v/v). Relative amounts of DNA/nucleus were determined on Feulgen-stained nuclei from O-2 mm terminal meristems with a Leitz microspectrophotometer [2] with the use of the two-wavelength method of Ornstein [4] and Patau [5]. Some samples were examined with a Zeiss scanning microspectrophotometer at 560 nm. Both methods were comparable when DNA/ nucleus was normalized with readings of one-half telophase and prophase taken to be 2C and 4C values, respectively:
Results and Discussion
Specifically, we approached the problem of whether cotyledon presence and absence influenced cell arrest in three ways. The first experiments were performed to demonstrate reduced ceil arrest in G2 in older cultured primary root tips of Pisum satiuum. Excised l-l.5 cm root tips immediately starved of carbohydrate for 4 days, had terminal O-2 mm apices with 41 k3 % cells of a 2C nuclear amount and 59 & 3 % witha4Camount of DNA (fig. 1). In apices of root tips first cultured for 7 days in medium with 2% sucrose and then starved of carbohydrate for 5 days, 78 k 2 y; of the nuclei had a 2 C and 22 L 2 % had a 4C nuclear DNA content (fig. 2). The Exptl Ceil Res 82 (1973)
H. MIETTINEN
and L. SAXEN,
Third Department of Pathology and Department Zoology, University of Helsinki, Finland
of
Summary. Dactinomycin is strongly adsorbed to nitrocellulose filters and some of the adsorbed material is slowly released. This affects both the growth and development of embryonic explants and the nucleotide incorporation of HeLa cells cultivated on pretreated filters.
Nitrocellulose (Millipore) filters are widely used in experimental biology both for sterile filtration and for tissue culture work. We obtained varying results in experiments on the effect of dactinomycin treatment on embryonic tissues separated by these filters in vitro cultures. This suggestedthat dactinomycin was adsorbed to the filter, then slowly releasedand incorporated in the tissue. Hence experiments were designed to investigate this. Material and Methods In transfilter experiments embryonic metanephric mesenchvme and spinal cord were cemented on both sides of a “TA” Millipore filter and cultured for 72 h in Eagle’s Minimum Essential Medium (MEM) supplemented with 10 % fetal calf serum (FCS) [l, 2,4]. Viability and differentiation was evaluated by light microscopy of serially sectioned explants. Filters were treated with SH-dactinomvcin in MEM containing 10 11 FCS for 1 h at 37”C, and then rinsed 3 times. For autoradiography explants were fixed in Carnoy’s solution, dehydrated in ethanol and cleared with benzene, embedded in paraffine and sectioned. Kodak AR 10 stripping film was used. For the evaluation of adsorption and release of dactinomycin, 1 cm2 pieces of 25 pm thick nitrocellulose filters (type TA, Millipore, Bedford, Mass.) were placed in test tubes containing 1 ml 3H-dactinomycin (Actinomycin D (aH), spec. act. 3.7 Ci/mmol; Schwarz/ Mann. Oranaeburg. N.Y.) in MEM for 30 min. Filters were -then -rinsed 3 times with phosphatebuffered saline (PBS). The radioactivitv was determined by liquid scintillation spectrometry. Five ml of cultivation medium containing 10 % FCS and 0.04 nmole/ml SH-dactinomycin or serum-free medium with 0.04 nmoleiml 8H-dactinomvcin and 0.36 nmoIe/ml non-radioactive dactinomycin were sterilized by filtration through a 13 mm diameter, 150 ,urn thick nitrocellulose filter (Millipore, type GS) fitted in a plastic holder (Swinnex, Millipore).
469
Twenty 1 cm2 pieces of TA filter were placed in 0.4 ,&i/ml SH-dactinomycin in 20 ml of PBS, incubated for 30 min and rinsed as above. One filter was placed in each tube with 2 ml MEM and incubated at 37°C. After 0 to 7 days, filters were removed and rinsed in PBS. Nitrocellulose filters were treated with dactinomycin in PBS for 30 min, rinsed 3 times with PBS and placed in 35 mm Petri dishes (Falcon Plastics, Oxnard, Calif.). Trypsinized HeLa cells were seeded onto these filters and cultivated for 3 days in MEM supplemented with 10% newborn calf serum. 3Huridine (NEN) was added to give a final concentration of 1 &i/ml. After 1 h the filters were removed and rinsed. The radioactivity was measured in half of the filters and the morphology of the cells was evaluated on the other half of the filters.
Results
A 1 cm2 filter with a volume of only about 0.0025 cm3and weighing about 1 mg removed 10-22~~ of the dactinomycin from a volume of 1.0 ml. More dactinomycin was removed from a serum-free than from a serumcontaining medium. The concentration of dactinomycin was 100-200 times greater in the filter than in the medium (table 1). About 5% of the dactinomycin adsorbed onto nitrocellulose filters was released during the first hour of incubation and about. 30% within one day with little change during further incubation. When a dactinornycincontaining solution was sterilized by filtration through a nitrocellulose filter there was a decrease in concentration, especially if the medium was serum-free (fig. 1). HeLa cells cultivated on filters treated with Table 1. Adsorption of dactinom,ycin to nitrocellulose ,filters Percentage removed by filter Initial cont. in medium (nmole/ ml) 0.004 0.04 0.4 4
Serumcontaining medium 13
16 II
10
Serumfree medium 15 19 22 14
Cont. in filter (nmole/g j1S.E.) --__ SerumconSerumtaining free medium medium 0.49 +0.04 0.54 to. I1 6.2 kO.5 7.01 0.6 45&10 83 f 1 380 & 30 580 4 30 -Exptl Cell Rrs HZ (1973)
470
Preliminary notes
I
2
3
4
5
Fig. 1. Abscissa: effluent volume from syringe, ml; ordinate: dactinomycin left in each ml of effluent, %. H, 0.4 nmole/ml in MEM; 0, 0.04 nmole/ml containing 10 % FCS. Amount of dactinomycin left after sterile filtration with nitrocellulose filters.
adsorbed to the filter seems to correlate fairly well with the concentration of dactinomycin in the medium. With the concentrations used no saturation effect was obtained. The adsorption of dactinomycin to nitrocellulose filters is quantitatively similar to that of protamine sulphate [3], but the mechanism of binding of dactinomycin to the filter is not known. The growth of both monolayer cells and tissue explants on dactinomycin-treated filters is severely affected. Embryonic tissues have been previously cultivated on nitrocellulose filters in the presence of dactinomycin [5, 61 and apparently, after removal of the dactinomycin, the filter has not been changed. It is difficult to evaluate whether this had any effect on the results of these experiments. Our conclusion is that whenever dactinomycin is used in embryological experiments
0.04 nmole/ml dactinomycin incorporated less 3H-UdR (1 600 cpm) than controls (8 400 cpm), and the morphology of the cells was poor. This experiment was repeated 3 times with qualitatively similar results. When spinal cord and metanephric mesenthyme were cultivated in a transfilter position on a 3H-dactinomycin-treated nitrocellulose filter there was no tubule formation (8 explants) in contrast to the tubule positive controls (9 explants). Autoradiograms showed grains on nuclei of both the spinal cord and of the mesenchyme (fig. 2). In these experiments the filters released so much dactinomycin that the medium concentration was 10 nCi/ml or 0.0027 nmole/ml. Discussion
The results indicate that dactinomycin is adsorbed to Millipore filters and that filter pieces can reduce the concentration of dactinomycin. Some of the adsorbed dactinomycin is released from the filter. The amount Exptl Cell Res 82 (1973)
Fig. 2. Uptake of 3H-dactinomycin by explants located on SH-dactinomycin-treated filter. Kodak AR 10 stripping film, exposure time 2 weeks, x 1 000. Spinal cord is located below and mesenchyme above the filter.
Preliminary notes
nitrocellulose filters have to be changed, since otherwise the duration of a pulse treatment may be longer than intended or, if the treatment is continuous, the local dactinomycin concentration may exceedthe bulk one. Furthermore, if diluted dactinomycin solutions are sterilized by filtration through nitrocellulose filters the final concentration may decreaseconsiderably. Grants: The Sigrid Juselius Foundation Foundation for Cancer Research, Finland.
and the
47 I
[2]. It is possible that fewer cells arrested in G2 in the older cultures becausea substance(s) was depleted in the root that is normally supplied by other plant organs. In the early stages of seedling growth the cotyledons are the chief source of most cell nutrients and consequently, they are most suspect as the tissue origin of the postulated substance(s) affecting cell arrest in G2. The experiments described in this paper were designed to test if the cotyledons influence cell arrest in G2 in root meristems of Pisum.
References 1. Grobstein, C, Exptl cell res 10 (1956) 424. 2. Jainchill, J, Saxen, L & Vainio, T, J embryo1 exptl morph01 12 (1964) 597. 3. Nordling, S, Miettinen, H, Wartiovaara, J & Saxen, L, J embryo1 exutl morph01 26 (1971) 231. 4. Sax& L & Sakseia, E, Exptl cell res 66 (1971) 349. 5. Wessells, N K, Devel biol 9 (1964) 92. 6. Wessells, N K & Wilt, F H, J mol biol 13 (1965) 767. Received June 25, 1973 Revised version received August 20, 1973
Cell arrest in G2 in root meristems:
A control factor from the cotyledons L. S. EVANS and J. VAN’T HOF, Biology Department, Brookhaven National Laboratory, Upton, N. Y. 11973, USA Summary. A substance promoting cell cycle arrest in G2 in the root meristem is demonstrated. This substance is produced in the cotyledons and is transported to the root.
Arrest in the mitotic cycle by meristematic cells in cultured primary root tips after protracted carbohydrate starvation is a nonrandom process [l]. Few, if any, cells stop during DNA synthesis and none arrest during mitosis. The probability of arrest in Gl and in G2 is not always equal. For example, in freshly excised carbohydrate-deficient meristems of Vicia faba the proportion of cells arrested, G 1 : G2, was approx. 1: 3 while that of older cultured meristems was about 1: 1
Materials and Methods The general procedures used in our experiments have been described previously [3]. Seeds of Pisum satiaum were surface sterilized in undiluted Clorox bleach containing 5.2 X sodium hypochlorite for 5 min, washed with water, and placed in sterile vermiculite. Three and 7 days after planting, l-l.5 cm primary root tips were placed into culture medium [6] with or without sucrose. Roots from seedlings were fixed after treatment in ethanol/acetic acid (3 : I, v/v). Relative amounts of DNA/nucleus were determined on Feulgen-stained nuclei from O-2 mm terminal meristems with a Leitz microspectrophotometer [2] with the use of the two-wavelength method of Ornstein [4] and Patau [5]. Some samples were examined with a Zeiss scanning microspectrophotometer at 560 nm. Both methods were comparable when DNA/ nucleus was normalized with readings of one-half telophase and prophase taken to be 2C and 4C values, respectively:
Results and Discussion
Specifically, we approached the problem of whether cotyledon presence and absence influenced cell arrest in three ways. The first experiments were performed to demonstrate reduced ceil arrest in G2 in older cultured primary root tips of Pisum satiuum. Excised l-l.5 cm root tips immediately starved of carbohydrate for 4 days, had terminal O-2 mm apices with 41 k3 % cells of a 2C nuclear amount and 59 & 3 % witha4Camount of DNA (fig. 1). In apices of root tips first cultured for 7 days in medium with 2% sucrose and then starved of carbohydrate for 5 days, 78 k 2 y; of the nuclei had a 2 C and 22 L 2 % had a 4C nuclear DNA content (fig. 2). The Exptl Ceil Res 82 (1973)