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Partial characterization of mosquito larvae extract modulating mouse and human cell proliferation
Cell Biology International 1999, Vol. 23, No. 3, 219–226 Article No. cbir.1998.0337, available online at http://www.idealibrary.com on
PARTIAL CHARACTERIZATION OF MOSQUITO LARVAE EXTRACT MODULATING MOUSE AND HUMAN CELL PROLIFERATION J. R. RONDEROS1*, O. J. RIMOLDI2, M. A. SALAS3 and R. R. BRENNER2 1
Ca´tedra de Histologı´a y Embriologı´a ‘B’, Facultad de Ciencias Me´dicas—Universidad Nacional de la Plata, Calle 60 y 120, 1900 La Plata, Argentina 2 INIBIOLP, UNLP-CONICET; and 3Ca´tedra de Fisiologı´a con Biofı´sica, FCM-UNLP, Argentina Received 20 April 1998, accepted 9 December 1998
Mosquito larvae crude extract have been found to alter the mitotic rate of several mouse epithelial cell populations such as enterocytes and tongue keratinocytes. Also, the dialysed fraction inhibits hepatocyte proliferation in hepatectomized males. These experiments suggested an inhibitory effect on the G1/S interphase. Consequently, we suggested the presence of some molecule or molecules related to the TGF- superfamily. In the present paper, we have assayed the crude extract on human mononuclear cells and the dialysed fraction of the extract on tongue keratinocyte proliferation. Furthermore, different protein fractions obtained using a molecular exclusion chromatographic column were assayed on hepatocyte proliferation of hepatectomized mice. Three groups of proteins have been isolated. Results show a dosedependent effect of crude extract on mononuclear cell proliferation and the dialysed extract caused an inhibitory effect on tongue keratinocyte proliferation. With regard to the hepatocyte mitotic rate, an inhibitory effect appeared only in animals receiving the fraction with lower molecular weight. These results suggest the presence in mosquito larvae of some peptidic molecule or molecules resembling the activity of members of the TGF- superfamily. 1999 Academic Press
K: keratinocytes; hepatocytes; mononuclear cells; mosquito; growth factors.
INTRODUCTION Cell populations have different cycle times which can vary according to distinct life times. Moreover, there exist cycling and noncycling cells in every population, depending on the requirements of the organism. Cell growth is regulated by several factors such as hormones, growth factors and cell surface receptor synthesis. As growth caused by cell proliferation is a fundamental process in biology, some genes involved in it have probably appeared in the genome by the time of mitosis appearance. Several factors controlling growth mechanisms such as growth factors *To whom correspondence should be addressed. E-mail: [email protected] The biological material used in the in vivo experiments performed in this paper was collected from Ca´tedera de Histologı´a y Embriologı´a ‘A’, Facultad de Ciencias Me´dicas, Universidad Nacional de La Plata, where Dr J. R. Ronderos developed his activities until July 1996. 1065–6995/99/030219+08 $30.00/0
and proto-oncogenes, have been maintained in nature throughout evolution. Thus, such distant organisms as insects and mammals share some of these genes. Genes codifying proteins structurally related to transforming growth factor-beta (TGF-) and epidermal growth factor (EGF) have been found in insects, nematodes and echinoderms (Padgett et al., 1987; Massague, 1990; Muskavitch and Hoffmann, 1990; Doctor et al., 1992; Ren et al., 1996). The presence of a gene in Drosophila that encodes a receptor of the TGF- superfamily was also evidenced (Tin Xie et al., 1994). Furthermore, the decapentaplegic gene, which codifies a protein highly homologous with TGF-, was reported as an imaginal disk cell synchroniser at the G2/M interphase during Drosophila eye development (Penton et al., 1997). In view of these facts, it could be expected that the mechanism controlling cell growth were not species specific. 1999 Academic Press
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On the other hand, it was demonstrated that in Aedes aegipty larvae there exist some cell populations which do not grow by hyperplasia (Tragger, 1937). This fact might be related to the existence of some molecule or molecules inhibiting cell proliferation and perhaps inducing cell differentiation. In order to find growth factors homologous of vertebrates in insects, we have assayed a crude extract from other mosquito larvae species (Culex pipiens L.) on the mitotic rate of several epithelial cell populations of mice in vivo. Thus, we have demonstrated that the crude extract of mosquito larvae has an inhibitory effect on the mitotic rate of hepatocytes of partially hepatectomized adult mice (Ronderos and Echave Llanos, 1990). The same effect was seen on hepatocytes, enterocytes, sialocytes and tongue keratinocytes of growing mice (Ronderos et al., 1994a; Ronderos et al., 1996). Similar results were found when the dialysed fraction of the extract (12 kDa cut-off pore membrane) was assayed on hepatocyte proliferation of hepatectomized adult mice, showing an association of the biological effect with the macromolecular fraction of the extract (Ronderos et al., 1994a). These results suggested the presence of some molecule resembling the activity of members of the TGFbeta superfamily or another unknown factor in mosquito larvae extract. In those experiments, treatments were applied before the DNA synthesis curve increased (Echave Llanos et al., 1971) and the sampling period was 16–24 h after treatment, when the mitotic peak occurs (Surur et al., 1985). The time elapsed between treatments and animal sacrifice suggested an inhibitory effect of the mosquito extract on the cell cycle G1/S checkpoint. When the mitotic rate-sampling period covered the dark phase of the circadian cycle (time of maximum DNA synthesis) a statistically significant increment was found in hepatocytes, nephrocytes and tongue keratinocytes of extract recipients. Taking into account the time elapsed from treatment administration until sacrifice (from 0–12 h), these results suggested an effect of the extract at the G2/M checkpoint of the cell cycle (Ronderos, 1996). In other experiments, treatments were applied after the DNA synthesis peak occurrence. The results were similar, showing decreased mitotic rates in hepatocytes, tongue keratinocytes and enterocytes (Ronderos et al., 1994b). The aim of the present study has been to assay both the dialysed extract on keratinocyte and three different protein fractions on hepatocyte proliferation of hepatectomized adult mice. Furthermore, taking into account that members of the TGF- superfamily have been also related to lymphocyte
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function by different authors (Ahuja et al., 1993; Altiok et al., 1994; De Jong et al., 1994; Fox et al., 1992; Fox et al., 1993; Lee and Rich, 1993; Reinhold et al., 1994), we decided to assay the effect of a crude preparation of mosquito larvae on the proliferation of human mononuclear cells (MNC). MATERIALS AND METHODS Mosquito larvae extract Third and fourth instar larvae of mosquito Culex pipiens L. (Diptera: Culicidae) were obtained from an artificial colony. Larvae were separated from the colony, washed several times in fresh water to eliminate any breed plate residue, then filtered and weighed for extract preparation. Pooled mosquito larvae were homogenized in a PotterDual homogenizer in an ice bath and saline solution was added (2:10) at 4C. The homogenate was centrifuged at 0C at 14,500g for 20 min in a Sorvall RC5C centrifuge and then dialysed with a 12,000 Dalton cut-off pore membrane in saline solution overnight at 4C. Gel permeation chromatography The dialysed homogenate was applied to a Superose 6HR 10/30 column, equilibrated and eluted with buffer Tris-HCl 10 m, ClNa 0.15 , pH 8.0 at a flow rate of 0.5 ml/min. The effluent was monitored at 280 nm. The protein-containing fractions were pooled and concentrated by ultrafiltration (Centricom-10, Amicon, Bedford). The protein contents were determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. Partially hepatectomized adult male mice C3HS (from Wilson in 1968) adult male mice (90 d old) were standardized for periodicity analysis (Echave Llanos and Nash, 1971; Halberg et al., 1958; Vilchez and Echave Llanos, 1971), single caged after weaning in a room ad hoc, lighted (fluorescent, 40 W) from 06:00–18:00 alternating with 12 h of darkness. Food and water were available ad libitum and the temperature was 221C. Mice experimental design C3HS male mice (90 d old) standardized for periodicity analysis for three weeks before surgery,
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Mitotic rate evaluation Tongues and the triangular lobe of the livers from experimental mice receiving either buffer saline solution, dialysed extract or protein fractions, were removed and processed for histological analysis (Echave Llanos and Sadnik, 1964). The mitotic rate was determined in histological slides by counting the colchicine-arrested metaphases in approximately 5000 cells at a magnification of 1000. Colcichine-arrested metaphases were recorded in every microscopic field and the total number of nuclei was counted every ten fields (liver). In the case of epithelial cells from the tongue, the mitotic rate was determined by counting the colchicinearrested metaphases of dorsal and ventral surfaces separately. Finally, mitotic rates were expressed as colchicine metaphases/1000 nuclei. Mononuclear cells isolation Human heparinized blood was obtained from healthy adult donors. MNC were isolated by centrifugation on a Ficoll gradient (Histopaque 1077-Sigma). After washing, cells were resuspended in RPMI medium supplemented with 2 m glutamine, 100 g/ml streptomycin, 100 U/ml penicillin, 2-ME 10 5 (all from Sigma) and 10% fetal bovine serum (Gen-Argentina), and then seeded into 96 well plates at a density of 105 cells/well (3 to 6 wells/treatment). MNC viability was evaluated by trypan blue exclusion test.
14 000
* 12 000
10 000
8000 cpm
were partially hepatectomized (70%) at 12:00 h. After 28 h (i.e. 16/28 time of day/time post hepatectomy (TD/TPH)) the animals were intraperitoneally injected (left side), killed by decapitation and exsanguination at 08/44; 12/48 and 16/52 (dialysed fraction on tongue keratinocyte proliferation). On the other hand, for the regeneratingliver assay fractions, mice were killed at 12/48 TD/TPH (time of mitotic peak). All animals received 0.01 ml of the different solutions by gram of body weight. As regards treatments, saline and dialysed larvae extracts (2.2 mg/ml) were assayed for tongue keratinocyte proliferation. For regenerating-liver experiments, the treatments assayed were the following: saline solution as general control of the experiment, dialysed fraction (2.0 mg/ml) as mosquito larvae control effect and chromatography eluted fractions (F1: 1.4 mg/ml; F2: 1.0 mg/ml; F3: 0.8 mg/ml). Colchicine (2.0 g/g body weight in 0.01 ml of distilled water) was also injected intraperitoneally (left side) to all animals 4 h before death.
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6000
* 4000
2000
* 0
Ctrl
1
0.5 0.1 Doses (mg/ml)
0.05
0.01
Fig. 1. Dose-dependent proliferative response of ConA activated MNC to mosquito crude extract. Bars represent mean.. Ctrl: controls without extract. *Statistically significant differences, P<0.05.
MNC experimental design MNC were cultured for 72 h at 37C in a humidified atmosphere of 5% CO2 in air. The different treatments and the mitogen Concanavalin A (ConA, 5 g/ml) diluted in RPMI were added simultaneously at the beginning of the culture. In order to evaluate lymphocyte proliferation [3H]Thymidine (1 Ci/well) was added for the last 4 h of culture. Cells were finally recovered on filters by the use of a cell harvester (Titerteck Cell Harvester, Flow Lab, U.S.A.). The radioactivity incorporated was measured in a liquid scintillation counter. Statistical analysis The experimental design for tongue keratinocyte assay involves variations in the mitotic rate produced by both circadian rhythms and by differential proliferation of the dorsal and ventral surfaces (Ronderos et al., 1994b). Consequently, the differences were analysed by multifactorial analysis of variance, including three factors acting on the
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increment of the MNC entering into the S-phase of the cycle. Lower doses did not cause any effect (Fig. 1).
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Effect of temperature on biological activity of the extract
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With the aim of knowing the effect of temperature on the biological activity of the extract, it was heated at 90C for 30 min and then centrifuged at 1500 rpm for 5 min. The experimental design was similar to the one previously used. A treatment of 0.5 mg/ml was included as control of the inhibitory effect of the extract. Results showed that after heating the biological effect of the extract disappeared (Fig. 2, Table 1).
cpm
8000
6000
4000
* 2000
Dialysed fraction effect on tongue keratinocyte proliferation
0
Fresh
90°C Treatments
Ctrl
Fig. 2. Effect of fresh (0.5 mg/ml) and heated extract (90C for 30 min) on MNC proliferation. Bars represent means.. *Statistically significant difference.
mitotic rate of this cell population (i.e., treatments, sampling time and tongue surface). For hepatocyte proliferation as well as for human MNC assay statistical analyses were performed by one way analysis of variance. RESULTS Effects of the crude extract on MNC proliferation Doses of the crude extract ranging from 1 to 0.01 mg/ml were applied to ConA activated MNC at the beginning of the culture. [3H]Thymidine incorporation showed a dual response of MNC to the extract. Doses of 1.0 and 0.5 mg/ml caused a statistically significant decrease in proliferation, while 0.05 mg/ml of the extract led to a significant
The results showed, as in regenerating-liver (Ronderos et al., 1994a), a significant (P=0·001) decrease of the mitotic rate in tongue keratinocytes of dialysed extract-receiving mice when analysis included all factors (saline: 15.081.74, n=26; dialysed: 6.901.63, n=30) (Table 2). When the mitotic rate was analysed discriminating the effect of the sampling time, we found that differences were evident only at 12/48 TD/TPH (P=0.0001) (saline: 26.343.41, n=10; dialysed: 6.22.24, n=10; percentage of variation: 76.46) (Fig. 3). The analysis of treatment effects, discriminating between ventral and dorsal surface response, showed that both keratinocyte subpopulations underwent a statistically significant decrease in the mitotic rate of dialysed fraction-receiving mice (dorsal: P=0.02; ventral: P=0.005) (Fig. 4). Analysis of chromatographic fractions The mosquito larvae dialysed extract was applied to a gel permeation Superose 6B column and eluted with Tris-HCl saline buffer. Three main peaks were detected (Fig. 5). The first one (F1), with
Table 1. One way analysis of variance comparisons for ConA induced MNC proliferation after treatment with fresh (0.5 mg/ml) or 90C heated extract against controls only receiving ConA Source of variation
Sum of squares
DF
Mean squares
F-ratio
Treatment Residuals Total
2.5108 8.0106 2.6108
2 8 10
1.2108 1.0106 —
125.67 — —
P<0.0001 — —
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Table 2. Multifactorial analysis of variance comparisons for mitotic rates of tongue keratinoytes. Primary sources of variations were: (A) tongue surface; (B) sample time after treatments and (C) treatments performed (saline solution and dialysed extract) Source of variation A: Surface B: Sample Time C: Treatment Interactions AB AC BC ABC Residual Total
Sum of squares
DF
Mean squares
F-ratio
16.83 818.97 913.77
1 2 1
16.83 409.49 913.77
0.22 5.25 11.72
P=0.65 P<0.01 P=0.001
54.41 92.19 1409.38 13.75 3429.33 6772.63
2 1 2 2 44 55
27.21 92.19 704.69 6.87 77.94 —
0.35 1.18 9.04 0.09 — —
P=0.71 P<0.28 P=0.0005 P=0.92 — —
higher molecular weight, included the microsomal fraction. With the aim of eliminating and confirming the microsomal nature of F1, the extract was ultracentrifuged at 100,000g for 1 h. The chromatographic profile of the supernatant after
ultracentrifugation showed no presence of the F1 fraction. When the pellet was resuspended and eluted in the same chromatographic system, the F1 fraction appeared clearly defined (data not shown). 40
35
35
30
30
20
15
*
*
Mitosis/1000 nuclei
Mitosis/1000 nuclei
25
25
20
15
*
*
10
10 5
5 0
08/44
12/48
16/52 Treatments
Sal
Dial
Fig. 3. Mitotic rate every 4 h and for the whole sample period for tongue keratinocytes in adult hepatectomized mice. Data represent mean.. (*), Statistically significant differences. Sal: Mitotic rate of saline receptors from 08/44–16/52 TD/ TPH; Dial: Mitotic rate of dialysed extract receivers for the same sample period. Key: ——, dialysed; ——, saline.
0
Dorsal
Ventral
Fig. 4. Tongue keratinocyte mitotic rate at 12/48 TD/TPH. Effect of the dialysed fraction of the extract on dorsal and ventral epithelia of the tongue. Bars represent mean.. of the sample. (*), Stastically significant differences. Key: , saline; , dialysed.
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F3 80
F1
Relative absorbance at 280 nm
Mitosis/1000 nuclei
60
40
*
*
20
F2
0
Sal
Dial F2 Treatments
F3
Fig. 6. Comparison between hepatocyte mitotic rates in mice receiving saline, dialysed extract, F2 and F3 at 12/48 TD/TPH. Bars represent mean.. *Statistically significant differences.
On the contrary, a stimulating but not statistically significant effect (Tukey multiple range test) on the mitotic rate of hepatocytes appeared associated to the F2 fraction (Fig. 6) (Table 3). 10
20 30 40 Retention time (min)
50
Fig. 5. Chromatographic profile of mosquito larvae extract eluted through a Superose 6 HR 10/30 molecular exclusion column.
Effect on hepatocyte proliferation The first group of proteins (F1 in Fig. 5) included the microsomal fraction of the extract. This fraction showed no effect on the hepatocyte proliferation as no differences between mitotic rates for this group and saline solution-receiving mice were found (data not shown). A second experiment involved the other two isolated chromatographic fractions (F2 and F3 in Fig. 5). As above described, previous experiments showed an inhibitory effect of the crude and dialysed fraction of the mosquito larvae extract on hepatocyte proliferation. In this experiment F3 showed a similar inhibitory effect (saline: 42.437.18 n=6; F3: 10.652.58 n=5; P<0·005; percentage of variation: 74.9) (Fig. 6; Table 3).
Molecular weight determination of F3 fraction The molecular weight range of F3 soluble fraction was estimated by gel permeation chromatography on a Superdex HR200 column. Using linear regression analysis of the data points, a plot of log molecular weight versus relative mobility was constructed from known standards. The F3 fraction has a molecular weight range from 10–80 kDa with a maximum relative absorbance at 280 nm peaks at a molecular weight level of 25 kDa. DISCUSSION As we have shown, a significant alteration in the proliferation rate of both mouse keratinocytes and hepatocytes and human MNC appears. We have previously demonstrated that the mitotic rate of tongue keratinocytes in young growing mice follows a circadian rhythm pattern with a mitotic peak at 12:00 (Ronderos et al.,
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Table 3. One way analysis of variance comparisons for mitotic rates of hepatocytes between saline, dialysed, F2 and F3 receiving mice showing general statistically significant difference between treatments Source of variation
Sum of squares
DF
Mean squares
F-ratio
Treatment Residuals Total
10631.03 8438.49 19069.52
3 18 21
3543.68 468.81 —
7.56 — —
1996). Regarding the necessary delay existing between S-phase and mitosis, the peak of proliferation is coincident with the circadian variation previously described for the DNA synthesis in the same cell populations of mice (Scheving et al., 1978; Scheving et al., 1979). Since the DNA synthesis peak occurs around 04:00, the decrease of mitotic rate at 12/48 TD/TPH in dialysed extractreceiving mice (also significant for the whole sample period) suggests, as in hepatocytes and other cell populations previously analysed, an inhibitory effect of the extract at G1/S restriction point of the cell cycle. This effect could be related to some macromolecule with a molecular weight higher than 12 kD. Since F3 also showed an inhibitory effect on hepatocyte proliferation, experiments tending to analyse the effect of this fraction on mouse tongue keratinocytes are now being done. Regarding chromatographic fraction assays on liver-regenerating hepatocytes and in coincidence with our previous reports (Ronderos et al., 1994a), an association of the extract inhibitory effect with macromolecules was found. This effect was associated to the soluble fraction with the lowest molecular weight (F3) (estimated molecular weight range from 10–80 kD). By taking into account the time chosen to administrate the treatments (16/28 TD/ TPH, time of raising of DNA synthesis curve), the results suggest an inhibitory effect of the extract at the G1/S checkpoint of the cell cycle. The experiments performed on human MNC proliferation demonstrated a biphasic effect of the crude extract. There were both an inhibitory and a stimulatory effect depending on the doses applied. No effect was seen with the lowest dose used. Furthermore, since MNC proliferation was evaluated by [3H]-thymidine incorporation, an alteration in the number of cells entering in the S-phase was confirmed. The fact that the biological effect of the extract on these cells disappeared after heating at 90C for 30 min, could suggest a protein nature of the molecule or molecules involved.
P=0.02 — —
TGF-, which inhibits cell proliferation acting on late G1-phase cyclin-dependent kinases (Reddy et al., 1994) and other members of the family, have been suggested as mediators for the regulation of DNA synthesis and differentiation in both hepatocytes (Kay and Fausto, 1997; Russell et al., 1988; Michalopoulos, 1992; Michalopoulos and DeFrances, 1997) and lymphocytes (Ahuja et al., 1993; Altiok et al., 1994; De Jong et al., 1994; Fox et al., 1992; Fox et al., 1993; Lee and Rich, 1993; Reinhold et al., 1994). Members of this growth factor superfamily have been also related to the regulation of the endocrine function, particularly with respect to pituitary hormones (De et al., 1996; Jin et al., 1997; Murata and Ying, 1991; Pastorcic et al., 1995). We have shown that the dialysed extract from mosquito larvae also has a specific modulatory effect on the rat pituitary hormones release. It inhibits both prolactin and GH and stimulates LH and TSH release (unpublished results). Finally, our results suggest the existence of some known or unknown peptidic molecule modulating cell behaviour in mammals.
ACKNOWLEDGEMENTS Thanks are due to Mrs Rubini Crabouras and Mrs Gabriela Simonetto for her skilful technical assistance and to the members of the Insect Pathology Laboratory (CEPAVE-UNLP) for the mosquito larvae supply. REFERENCES A SS, P F, Y H, B JE, B DT, 1993. Effect of transforming growth factor-beta on early and late activation events in human T-cells. J Immunol 150(8 part 1): 3109–3118. A A, J M, M T, K E, 1994. Biphasic effect of transforming growth factor-beta on
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PARTIAL CHARACTERIZATION OF MOSQUITO LARVAE EXTRACT MODULATING MOUSE AND HUMAN CELL PROLIFERATION J. R. RONDEROS1*, O. J. RIMOLDI2, M. A. SALAS3 and R. R. BRENNER2 1
Ca´tedra de Histologı´a y Embriologı´a ‘B’, Facultad de Ciencias Me´dicas—Universidad Nacional de la Plata, Calle 60 y 120, 1900 La Plata, Argentina 2 INIBIOLP, UNLP-CONICET; and 3Ca´tedra de Fisiologı´a con Biofı´sica, FCM-UNLP, Argentina Received 20 April 1998, accepted 9 December 1998
Mosquito larvae crude extract have been found to alter the mitotic rate of several mouse epithelial cell populations such as enterocytes and tongue keratinocytes. Also, the dialysed fraction inhibits hepatocyte proliferation in hepatectomized males. These experiments suggested an inhibitory effect on the G1/S interphase. Consequently, we suggested the presence of some molecule or molecules related to the TGF- superfamily. In the present paper, we have assayed the crude extract on human mononuclear cells and the dialysed fraction of the extract on tongue keratinocyte proliferation. Furthermore, different protein fractions obtained using a molecular exclusion chromatographic column were assayed on hepatocyte proliferation of hepatectomized mice. Three groups of proteins have been isolated. Results show a dosedependent effect of crude extract on mononuclear cell proliferation and the dialysed extract caused an inhibitory effect on tongue keratinocyte proliferation. With regard to the hepatocyte mitotic rate, an inhibitory effect appeared only in animals receiving the fraction with lower molecular weight. These results suggest the presence in mosquito larvae of some peptidic molecule or molecules resembling the activity of members of the TGF- superfamily. 1999 Academic Press
K: keratinocytes; hepatocytes; mononuclear cells; mosquito; growth factors.
INTRODUCTION Cell populations have different cycle times which can vary according to distinct life times. Moreover, there exist cycling and noncycling cells in every population, depending on the requirements of the organism. Cell growth is regulated by several factors such as hormones, growth factors and cell surface receptor synthesis. As growth caused by cell proliferation is a fundamental process in biology, some genes involved in it have probably appeared in the genome by the time of mitosis appearance. Several factors controlling growth mechanisms such as growth factors *To whom correspondence should be addressed. E-mail: [email protected] The biological material used in the in vivo experiments performed in this paper was collected from Ca´tedera de Histologı´a y Embriologı´a ‘A’, Facultad de Ciencias Me´dicas, Universidad Nacional de La Plata, where Dr J. R. Ronderos developed his activities until July 1996. 1065–6995/99/030219+08 $30.00/0
and proto-oncogenes, have been maintained in nature throughout evolution. Thus, such distant organisms as insects and mammals share some of these genes. Genes codifying proteins structurally related to transforming growth factor-beta (TGF-) and epidermal growth factor (EGF) have been found in insects, nematodes and echinoderms (Padgett et al., 1987; Massague, 1990; Muskavitch and Hoffmann, 1990; Doctor et al., 1992; Ren et al., 1996). The presence of a gene in Drosophila that encodes a receptor of the TGF- superfamily was also evidenced (Tin Xie et al., 1994). Furthermore, the decapentaplegic gene, which codifies a protein highly homologous with TGF-, was reported as an imaginal disk cell synchroniser at the G2/M interphase during Drosophila eye development (Penton et al., 1997). In view of these facts, it could be expected that the mechanism controlling cell growth were not species specific. 1999 Academic Press
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On the other hand, it was demonstrated that in Aedes aegipty larvae there exist some cell populations which do not grow by hyperplasia (Tragger, 1937). This fact might be related to the existence of some molecule or molecules inhibiting cell proliferation and perhaps inducing cell differentiation. In order to find growth factors homologous of vertebrates in insects, we have assayed a crude extract from other mosquito larvae species (Culex pipiens L.) on the mitotic rate of several epithelial cell populations of mice in vivo. Thus, we have demonstrated that the crude extract of mosquito larvae has an inhibitory effect on the mitotic rate of hepatocytes of partially hepatectomized adult mice (Ronderos and Echave Llanos, 1990). The same effect was seen on hepatocytes, enterocytes, sialocytes and tongue keratinocytes of growing mice (Ronderos et al., 1994a; Ronderos et al., 1996). Similar results were found when the dialysed fraction of the extract (12 kDa cut-off pore membrane) was assayed on hepatocyte proliferation of hepatectomized adult mice, showing an association of the biological effect with the macromolecular fraction of the extract (Ronderos et al., 1994a). These results suggested the presence of some molecule resembling the activity of members of the TGFbeta superfamily or another unknown factor in mosquito larvae extract. In those experiments, treatments were applied before the DNA synthesis curve increased (Echave Llanos et al., 1971) and the sampling period was 16–24 h after treatment, when the mitotic peak occurs (Surur et al., 1985). The time elapsed between treatments and animal sacrifice suggested an inhibitory effect of the mosquito extract on the cell cycle G1/S checkpoint. When the mitotic rate-sampling period covered the dark phase of the circadian cycle (time of maximum DNA synthesis) a statistically significant increment was found in hepatocytes, nephrocytes and tongue keratinocytes of extract recipients. Taking into account the time elapsed from treatment administration until sacrifice (from 0–12 h), these results suggested an effect of the extract at the G2/M checkpoint of the cell cycle (Ronderos, 1996). In other experiments, treatments were applied after the DNA synthesis peak occurrence. The results were similar, showing decreased mitotic rates in hepatocytes, tongue keratinocytes and enterocytes (Ronderos et al., 1994b). The aim of the present study has been to assay both the dialysed extract on keratinocyte and three different protein fractions on hepatocyte proliferation of hepatectomized adult mice. Furthermore, taking into account that members of the TGF- superfamily have been also related to lymphocyte
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function by different authors (Ahuja et al., 1993; Altiok et al., 1994; De Jong et al., 1994; Fox et al., 1992; Fox et al., 1993; Lee and Rich, 1993; Reinhold et al., 1994), we decided to assay the effect of a crude preparation of mosquito larvae on the proliferation of human mononuclear cells (MNC). MATERIALS AND METHODS Mosquito larvae extract Third and fourth instar larvae of mosquito Culex pipiens L. (Diptera: Culicidae) were obtained from an artificial colony. Larvae were separated from the colony, washed several times in fresh water to eliminate any breed plate residue, then filtered and weighed for extract preparation. Pooled mosquito larvae were homogenized in a PotterDual homogenizer in an ice bath and saline solution was added (2:10) at 4C. The homogenate was centrifuged at 0C at 14,500g for 20 min in a Sorvall RC5C centrifuge and then dialysed with a 12,000 Dalton cut-off pore membrane in saline solution overnight at 4C. Gel permeation chromatography The dialysed homogenate was applied to a Superose 6HR 10/30 column, equilibrated and eluted with buffer Tris-HCl 10 m, ClNa 0.15 , pH 8.0 at a flow rate of 0.5 ml/min. The effluent was monitored at 280 nm. The protein-containing fractions were pooled and concentrated by ultrafiltration (Centricom-10, Amicon, Bedford). The protein contents were determined by the method of Lowry et al. (1951) using bovine serum albumin as standard. Partially hepatectomized adult male mice C3HS (from Wilson in 1968) adult male mice (90 d old) were standardized for periodicity analysis (Echave Llanos and Nash, 1971; Halberg et al., 1958; Vilchez and Echave Llanos, 1971), single caged after weaning in a room ad hoc, lighted (fluorescent, 40 W) from 06:00–18:00 alternating with 12 h of darkness. Food and water were available ad libitum and the temperature was 221C. Mice experimental design C3HS male mice (90 d old) standardized for periodicity analysis for three weeks before surgery,
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Mitotic rate evaluation Tongues and the triangular lobe of the livers from experimental mice receiving either buffer saline solution, dialysed extract or protein fractions, were removed and processed for histological analysis (Echave Llanos and Sadnik, 1964). The mitotic rate was determined in histological slides by counting the colchicine-arrested metaphases in approximately 5000 cells at a magnification of 1000. Colcichine-arrested metaphases were recorded in every microscopic field and the total number of nuclei was counted every ten fields (liver). In the case of epithelial cells from the tongue, the mitotic rate was determined by counting the colchicinearrested metaphases of dorsal and ventral surfaces separately. Finally, mitotic rates were expressed as colchicine metaphases/1000 nuclei. Mononuclear cells isolation Human heparinized blood was obtained from healthy adult donors. MNC were isolated by centrifugation on a Ficoll gradient (Histopaque 1077-Sigma). After washing, cells were resuspended in RPMI medium supplemented with 2 m glutamine, 100 g/ml streptomycin, 100 U/ml penicillin, 2-ME 10 5 (all from Sigma) and 10% fetal bovine serum (Gen-Argentina), and then seeded into 96 well plates at a density of 105 cells/well (3 to 6 wells/treatment). MNC viability was evaluated by trypan blue exclusion test.
14 000
* 12 000
10 000
8000 cpm
were partially hepatectomized (70%) at 12:00 h. After 28 h (i.e. 16/28 time of day/time post hepatectomy (TD/TPH)) the animals were intraperitoneally injected (left side), killed by decapitation and exsanguination at 08/44; 12/48 and 16/52 (dialysed fraction on tongue keratinocyte proliferation). On the other hand, for the regeneratingliver assay fractions, mice were killed at 12/48 TD/TPH (time of mitotic peak). All animals received 0.01 ml of the different solutions by gram of body weight. As regards treatments, saline and dialysed larvae extracts (2.2 mg/ml) were assayed for tongue keratinocyte proliferation. For regenerating-liver experiments, the treatments assayed were the following: saline solution as general control of the experiment, dialysed fraction (2.0 mg/ml) as mosquito larvae control effect and chromatography eluted fractions (F1: 1.4 mg/ml; F2: 1.0 mg/ml; F3: 0.8 mg/ml). Colchicine (2.0 g/g body weight in 0.01 ml of distilled water) was also injected intraperitoneally (left side) to all animals 4 h before death.
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6000
* 4000
2000
* 0
Ctrl
1
0.5 0.1 Doses (mg/ml)
0.05
0.01
Fig. 1. Dose-dependent proliferative response of ConA activated MNC to mosquito crude extract. Bars represent mean.. Ctrl: controls without extract. *Statistically significant differences, P<0.05.
MNC experimental design MNC were cultured for 72 h at 37C in a humidified atmosphere of 5% CO2 in air. The different treatments and the mitogen Concanavalin A (ConA, 5 g/ml) diluted in RPMI were added simultaneously at the beginning of the culture. In order to evaluate lymphocyte proliferation [3H]Thymidine (1 Ci/well) was added for the last 4 h of culture. Cells were finally recovered on filters by the use of a cell harvester (Titerteck Cell Harvester, Flow Lab, U.S.A.). The radioactivity incorporated was measured in a liquid scintillation counter. Statistical analysis The experimental design for tongue keratinocyte assay involves variations in the mitotic rate produced by both circadian rhythms and by differential proliferation of the dorsal and ventral surfaces (Ronderos et al., 1994b). Consequently, the differences were analysed by multifactorial analysis of variance, including three factors acting on the
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increment of the MNC entering into the S-phase of the cycle. Lower doses did not cause any effect (Fig. 1).
14 000
12 000
Effect of temperature on biological activity of the extract
10 000
With the aim of knowing the effect of temperature on the biological activity of the extract, it was heated at 90C for 30 min and then centrifuged at 1500 rpm for 5 min. The experimental design was similar to the one previously used. A treatment of 0.5 mg/ml was included as control of the inhibitory effect of the extract. Results showed that after heating the biological effect of the extract disappeared (Fig. 2, Table 1).
cpm
8000
6000
4000
* 2000
Dialysed fraction effect on tongue keratinocyte proliferation
0
Fresh
90°C Treatments
Ctrl
Fig. 2. Effect of fresh (0.5 mg/ml) and heated extract (90C for 30 min) on MNC proliferation. Bars represent means.. *Statistically significant difference.
mitotic rate of this cell population (i.e., treatments, sampling time and tongue surface). For hepatocyte proliferation as well as for human MNC assay statistical analyses were performed by one way analysis of variance. RESULTS Effects of the crude extract on MNC proliferation Doses of the crude extract ranging from 1 to 0.01 mg/ml were applied to ConA activated MNC at the beginning of the culture. [3H]Thymidine incorporation showed a dual response of MNC to the extract. Doses of 1.0 and 0.5 mg/ml caused a statistically significant decrease in proliferation, while 0.05 mg/ml of the extract led to a significant
The results showed, as in regenerating-liver (Ronderos et al., 1994a), a significant (P=0·001) decrease of the mitotic rate in tongue keratinocytes of dialysed extract-receiving mice when analysis included all factors (saline: 15.081.74, n=26; dialysed: 6.901.63, n=30) (Table 2). When the mitotic rate was analysed discriminating the effect of the sampling time, we found that differences were evident only at 12/48 TD/TPH (P=0.0001) (saline: 26.343.41, n=10; dialysed: 6.22.24, n=10; percentage of variation: 76.46) (Fig. 3). The analysis of treatment effects, discriminating between ventral and dorsal surface response, showed that both keratinocyte subpopulations underwent a statistically significant decrease in the mitotic rate of dialysed fraction-receiving mice (dorsal: P=0.02; ventral: P=0.005) (Fig. 4). Analysis of chromatographic fractions The mosquito larvae dialysed extract was applied to a gel permeation Superose 6B column and eluted with Tris-HCl saline buffer. Three main peaks were detected (Fig. 5). The first one (F1), with
Table 1. One way analysis of variance comparisons for ConA induced MNC proliferation after treatment with fresh (0.5 mg/ml) or 90C heated extract against controls only receiving ConA Source of variation
Sum of squares
DF
Mean squares
F-ratio
Treatment Residuals Total
2.5108 8.0106 2.6108
2 8 10
1.2108 1.0106 —
125.67 — —
P<0.0001 — —
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Table 2. Multifactorial analysis of variance comparisons for mitotic rates of tongue keratinoytes. Primary sources of variations were: (A) tongue surface; (B) sample time after treatments and (C) treatments performed (saline solution and dialysed extract) Source of variation A: Surface B: Sample Time C: Treatment Interactions AB AC BC ABC Residual Total
Sum of squares
DF
Mean squares
F-ratio
16.83 818.97 913.77
1 2 1
16.83 409.49 913.77
0.22 5.25 11.72
P=0.65 P<0.01 P=0.001
54.41 92.19 1409.38 13.75 3429.33 6772.63
2 1 2 2 44 55
27.21 92.19 704.69 6.87 77.94 —
0.35 1.18 9.04 0.09 — —
P=0.71 P<0.28 P=0.0005 P=0.92 — —
higher molecular weight, included the microsomal fraction. With the aim of eliminating and confirming the microsomal nature of F1, the extract was ultracentrifuged at 100,000g for 1 h. The chromatographic profile of the supernatant after
ultracentrifugation showed no presence of the F1 fraction. When the pellet was resuspended and eluted in the same chromatographic system, the F1 fraction appeared clearly defined (data not shown). 40
35
35
30
30
20
15
*
*
Mitosis/1000 nuclei
Mitosis/1000 nuclei
25
25
20
15
*
*
10
10 5
5 0
08/44
12/48
16/52 Treatments
Sal
Dial
Fig. 3. Mitotic rate every 4 h and for the whole sample period for tongue keratinocytes in adult hepatectomized mice. Data represent mean.. (*), Statistically significant differences. Sal: Mitotic rate of saline receptors from 08/44–16/52 TD/ TPH; Dial: Mitotic rate of dialysed extract receivers for the same sample period. Key: ——, dialysed; ——, saline.
0
Dorsal
Ventral
Fig. 4. Tongue keratinocyte mitotic rate at 12/48 TD/TPH. Effect of the dialysed fraction of the extract on dorsal and ventral epithelia of the tongue. Bars represent mean.. of the sample. (*), Stastically significant differences. Key: , saline; , dialysed.
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F3 80
F1
Relative absorbance at 280 nm
Mitosis/1000 nuclei
60
40
*
*
20
F2
0
Sal
Dial F2 Treatments
F3
Fig. 6. Comparison between hepatocyte mitotic rates in mice receiving saline, dialysed extract, F2 and F3 at 12/48 TD/TPH. Bars represent mean.. *Statistically significant differences.
On the contrary, a stimulating but not statistically significant effect (Tukey multiple range test) on the mitotic rate of hepatocytes appeared associated to the F2 fraction (Fig. 6) (Table 3). 10
20 30 40 Retention time (min)
50
Fig. 5. Chromatographic profile of mosquito larvae extract eluted through a Superose 6 HR 10/30 molecular exclusion column.
Effect on hepatocyte proliferation The first group of proteins (F1 in Fig. 5) included the microsomal fraction of the extract. This fraction showed no effect on the hepatocyte proliferation as no differences between mitotic rates for this group and saline solution-receiving mice were found (data not shown). A second experiment involved the other two isolated chromatographic fractions (F2 and F3 in Fig. 5). As above described, previous experiments showed an inhibitory effect of the crude and dialysed fraction of the mosquito larvae extract on hepatocyte proliferation. In this experiment F3 showed a similar inhibitory effect (saline: 42.437.18 n=6; F3: 10.652.58 n=5; P<0·005; percentage of variation: 74.9) (Fig. 6; Table 3).
Molecular weight determination of F3 fraction The molecular weight range of F3 soluble fraction was estimated by gel permeation chromatography on a Superdex HR200 column. Using linear regression analysis of the data points, a plot of log molecular weight versus relative mobility was constructed from known standards. The F3 fraction has a molecular weight range from 10–80 kDa with a maximum relative absorbance at 280 nm peaks at a molecular weight level of 25 kDa. DISCUSSION As we have shown, a significant alteration in the proliferation rate of both mouse keratinocytes and hepatocytes and human MNC appears. We have previously demonstrated that the mitotic rate of tongue keratinocytes in young growing mice follows a circadian rhythm pattern with a mitotic peak at 12:00 (Ronderos et al.,
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Table 3. One way analysis of variance comparisons for mitotic rates of hepatocytes between saline, dialysed, F2 and F3 receiving mice showing general statistically significant difference between treatments Source of variation
Sum of squares
DF
Mean squares
F-ratio
Treatment Residuals Total
10631.03 8438.49 19069.52
3 18 21
3543.68 468.81 —
7.56 — —
1996). Regarding the necessary delay existing between S-phase and mitosis, the peak of proliferation is coincident with the circadian variation previously described for the DNA synthesis in the same cell populations of mice (Scheving et al., 1978; Scheving et al., 1979). Since the DNA synthesis peak occurs around 04:00, the decrease of mitotic rate at 12/48 TD/TPH in dialysed extractreceiving mice (also significant for the whole sample period) suggests, as in hepatocytes and other cell populations previously analysed, an inhibitory effect of the extract at G1/S restriction point of the cell cycle. This effect could be related to some macromolecule with a molecular weight higher than 12 kD. Since F3 also showed an inhibitory effect on hepatocyte proliferation, experiments tending to analyse the effect of this fraction on mouse tongue keratinocytes are now being done. Regarding chromatographic fraction assays on liver-regenerating hepatocytes and in coincidence with our previous reports (Ronderos et al., 1994a), an association of the extract inhibitory effect with macromolecules was found. This effect was associated to the soluble fraction with the lowest molecular weight (F3) (estimated molecular weight range from 10–80 kD). By taking into account the time chosen to administrate the treatments (16/28 TD/ TPH, time of raising of DNA synthesis curve), the results suggest an inhibitory effect of the extract at the G1/S checkpoint of the cell cycle. The experiments performed on human MNC proliferation demonstrated a biphasic effect of the crude extract. There were both an inhibitory and a stimulatory effect depending on the doses applied. No effect was seen with the lowest dose used. Furthermore, since MNC proliferation was evaluated by [3H]-thymidine incorporation, an alteration in the number of cells entering in the S-phase was confirmed. The fact that the biological effect of the extract on these cells disappeared after heating at 90C for 30 min, could suggest a protein nature of the molecule or molecules involved.
P=0.02 — —
TGF-, which inhibits cell proliferation acting on late G1-phase cyclin-dependent kinases (Reddy et al., 1994) and other members of the family, have been suggested as mediators for the regulation of DNA synthesis and differentiation in both hepatocytes (Kay and Fausto, 1997; Russell et al., 1988; Michalopoulos, 1992; Michalopoulos and DeFrances, 1997) and lymphocytes (Ahuja et al., 1993; Altiok et al., 1994; De Jong et al., 1994; Fox et al., 1992; Fox et al., 1993; Lee and Rich, 1993; Reinhold et al., 1994). Members of this growth factor superfamily have been also related to the regulation of the endocrine function, particularly with respect to pituitary hormones (De et al., 1996; Jin et al., 1997; Murata and Ying, 1991; Pastorcic et al., 1995). We have shown that the dialysed extract from mosquito larvae also has a specific modulatory effect on the rat pituitary hormones release. It inhibits both prolactin and GH and stimulates LH and TSH release (unpublished results). Finally, our results suggest the existence of some known or unknown peptidic molecule modulating cell behaviour in mammals.
ACKNOWLEDGEMENTS Thanks are due to Mrs Rubini Crabouras and Mrs Gabriela Simonetto for her skilful technical assistance and to the members of the Insect Pathology Laboratory (CEPAVE-UNLP) for the mosquito larvae supply. REFERENCES A SS, P F, Y H, B JE, B DT, 1993. Effect of transforming growth factor-beta on early and late activation events in human T-cells. J Immunol 150(8 part 1): 3109–3118. A A, J M, M T, K E, 1994. Biphasic effect of transforming growth factor-beta on
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