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Expression of human metallothionein-III confers protection against serumfree exposure of stably transfected Chinese hamster ovary CHO-K1 cells
ELSEVIER
Neuroscience
titters
201 (1995) 61-64
Expression of human metallothionein-III confers protection against serumfree exposure of stably transfected Chinese hamster ovary CHO-Kl cells Marie-Claude Luborutory
Amoureux, Thierry Wurch, Petrus J. Pauwels*
qf Cellulur and Molecular Neurobiology, Centre de Recherche Pierre Fabre, 17, avenue Jean Moulin, 81106 Castres Cddex, France Received 10 July 199.5;revised version received 16 October 1995; accepted 20 October 1995
Abstract The cloned human metallothionein (MT)-111 coding region tias permanently transfected in Chinese hamster ovary (CHO-Kl) cells in order to investigate the growth regulatory effects of this brain-specific protein. Reverse transcription-polymerase chain reaction (RTPCR) experiments demonstrated stable expression of MT-III mRNA in CHO-KVMT-III cells. Whereas in the presence of serum no differences were observed between the cell growth of both CHO-KUMT-III and CHO-Kl/pcDNA3-plasmid transfected cells, cell survival was attenuated differently in serum-free medium. CHO-Kl/MT-III cells were more resistant (40 + 8%) to serum deprivation than pcDNA3-plasmid transfected cells. Recovery of cell growth for both cell lines was obtained by supplementing serum (0.25%), although with a different
growth rate; half-maximal
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium
bromide (Mm)-conversion
was ob-
served between 5.1-5.7 and 4.2-4.5 days for CHO-KUpcDNA3 and CHO-Kl/MT-III cells, respectively. These results suggest a protective role for cloned human MT-III, besides its reported inhibitory and stimulatory effects on cell survival. Keywords:
Metallothionein-III (cloned, human); Chinese hamster ovary (CHO-Kl) cell line; Permanent transfection; Reverse transcription-polymerase chain reaction; Cell survival; Serum starvation; Growth factor
Modifications in the brain content of stimulatory (such as nerve growth factor and brain-derived neurotrophic factor) and inhibitory (such as metallothionein (MT)-III) growth factors of Alzheimer’s disease (AD) patients have been reported [4,9,17,22]. MT-III has been shown to be down-regulated in astrocytes of the frontal cortex of AD patients [2 1,221. The authors put forward that reduction in the content of this growth inhibitory factor may stimulate neurotrophic activity of susceptible neurons in the brain by massive sprouting, this latter a well-known feature of AD brain [2,6]. This hypothesis was supported by the observed enhanced neuronal cell survival of rat brain neuronal cultures in the presence of brain extract [5,22] or cerebrospinal fluid [16] of AD patients. Erickson et al. [5], furthermore, showed enhanced neurotrophic activity in frontal cortex of AD patients, not associated, however, with down-regulation of MT-III protein and mRNA. These authors concluded that MT-III-down-regulation is probably not an important pathogenic event in AD. MTIII appeared to be inhibitory on neuronal cell survival in * Corresponding author. Tel.: +33 63 71 42 54; fax: +33 63 71 42 99.
0304-3940/95/$09.50 SSDI
0304-3940(95)
the presence of brain extract. In contrast, neuronal survival was increased when MT-III was added to cultures without brain extract [5]. Therefore, it seems that MT-III is not solely a growth inhibitory factor but may also stimulate neuronal cell survival. The mechanisms involved in regulation of cell survival by MT-III are at present not elucidated. Cloning and sequencing of the human, mouse and rat MT-III genes revealed that it is a member of the MT gene family [8,15,21]. Whereas MT-I and MT-II genes are expressed in many tissues including the brain, MT-III expression is virtually brain-specific [8, 15,221. Even though MT-III is brain-specific, we were interested in the effect of MT-III on the survival and growth of a cell type that did not constitutively express MT-III. Therefore, the coding region of human MT-III was cloned and permanently transfected in Chinese hamster ovary (CHO-Kl) cells. The expression of MT-III mRNA was followed by reverse transcription-polymerase chain reaction (RT-PCR) while MT-III transfected cells were monitored for their survival and growth rate in comparison with a pcDNA3-plasmid transfected CHO-Kl cell line.
0 1995 Elsevier Science Ireland Ltd. All rights reserved 12 134-M
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M.-C. Amoureux et al. I Neuroscience Letters 201 (1995) 61-64
The human MT-III coding region was amplified from human cerebral cortical cDNA (Clontech, USA) using PCR. A sense primer including a Hind111 restriction site (S-AGTCAAGC’ITATGGACCCTGAGACCTGCCCCTGCCCT-3’) and a reverse primer with a BumHI restriction site (S-AGTCGGATCCTCACTGGCAGCAGCTGCACTTCTCTGC’ITC-3’) were designed to amplify 207 bp of human MT-III cDNA [ 151. The PCR program consisted of 25 repetitive cycles with a denaturing step at 94°C for 1 min, an annealing step at 60°C for 1 min and an extension step at 72°C for 1 min. A single PCR product (200 bp) was inserted in pcDNA3-plasmid (Invitrogen, USA) and sequenced by the dideoxynucleotide chain termination procedure. CHO-Kl cells (lo7 cells, ATCC CCL61) were permanently transfected with 10 pug of pcDNA3/MT-III-plasmid using a gene pulser transfection apparatus (Bio-Rad) at 250 mV and 250pF and grown in nutrient mixture Ham’s F12 supplemented with 10% heat-inactivated foetal calf serum and 1.25 mg/ml geneticin. RNA was extracted using a total RNA separator kit (Clontech, USA), treated with DNaseI (Stratagene, USA) and reverse transcribed into cDNA before PCR was performed as previously described [l]. The sense and reverse primers described above without restriction sites were used to amplify human MT-III cDNA. Control experiments were performed with pcDNA3/MT-III-plasmid, cDNA prepared from human temporal cortical brain tissue, and CHO-Kl cells permanently transfected with pcDNA3-plasmid. The specificity of the amplified PCR products was determined by Ear1 (New England Biolabs, USA) restriction (10 U/PCR reaction for 1 h at 37°C). Quantitative RT-PCR of human MT-III mRNA was compared with the constitutive mRNA /?-actin as previously described [ 11. To follow cell growth and survival of the CHO-Kl cell lines, cells were washed twice with PBS, trypsinised (0.25% trypsin, 15 min at 37”(Z), and plated at the indicated densities either in the absence or presence of serum in 0.25 ml of nutrient mixture Ham’s F12 supplemented with 5pglml transferrin, 30 nM selenium, 5.35 pg/ml linoleic acid, 20 nM progesterone, 1OOpM putrescine, 5 lug/ml insulin and 0.1% bovine serum albumin, in a 24well tissue culture plate. At different times, cell survival was quantified by measuring 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (M’IT) conversion as previously described [ 10,121 or measuring [3H]thymidine (1 j&i per well) incorporation. A high yield (90%) of clones of CHO-Kl cells permanently expressing the human MT-III coding region was obtained, as confirmed by RT-PCR; results are shown for one representative clone in Fig. 1A. PCR amplification of 0.2 ng of pcDNA3-plasmid containing the MT-III coding region or 10 ng of RT, DNase-treated human temporal cortical RNA yields a single band of approximately 200 bp (lanes 1, 3). A similar result was obtained with 25 ng of RT, DNase-treated CHO-Kl/MT-III RNA (lane
Fig. 1. Expression of human MT-III mRNA in stably transfected CHOKl/MT-III cells. (A) RNA from CHO-Kl/MT-III cells and post-mortem human brain temporal cortex was isolated, treated with DNaseI and reverse transcribed (RT) before PCR was performed with human MTIII primers. Lane M, 100 bp DNA ladder (Gibco Laboratories, UK); lane 1, 0.2 ng pcDNA3/MT-III; lane 2, Ear1 digestion (10 U) of PCRamplified pcDNA3/MT-III; lane 3, 10 ng RT, DNase-treated human temporal cortical RNA; lane 4, Ear1 digestion of PCR-amplified, RT, DNase-treated human temporal cortical RNA; lane 5, 200 ng DNasetreated human temporal cortical RNA; lane 6,25 ng RT, DNase-treated CHO-KlIMT-III RNA; lane 7, Ear1 digestion of PCR-amplified, RT, DNase-treated CHO-Kl/MT-III cellular RNA; lane 8, 500 ng DNasetreated CHO-KUMT-III RNA; lane 9,500 ng RT, DNase-treated CHOKU pcDNA3 RNA. (B) RNA of CHO-KlIMT-III cells was DNasetreated, reverse transcribed and analysed for MT-III and /I-actin mRNA expression at subcultures 1 (lane l), 5 (lane 2) and IO (lane 3). Quantitative PCR was performed on 4-16 ng, and 0,025-l ng of RT, DNasetreated RNA, with human MT-III and rat B-actin primers, respectively. The figure. corresponds to 8 ng RNA for MT-III and 0.05 ng RNA for B-actin.
6). The enzymatic digestion of these PCR products by Ear1 showed in each case two additional fragments of 119 bp and 88 bp (lanes 2, 4, 7). This further shows that the PCR product amplified from cDNA of the MT-III transfected CHO-Kl cell line is specific for human MTIII. No amplification was measured in samples of DNasetreated human temporal cortical (200 ng) and CHOKl/MT-III (500 ng) RNA without reverse transcriptase treatment (lanes 5, 8) and RT, DNase-treated RNA of CHO-Kl/pcDNA3 cells (500 ng, lane 9). The mRNA expression ratio of MT-III versus /I-actin was stable (between 1.1 and 1.2) for at least ten subcultures (Fig. 1B). The growth of CHO-Kl/pcDNA3 and CHO-Kl/MTIII cells was apparently not different in the presence of serum. This was obvious for various plating densities between 3000 and 150 000 cells per well, various concen-
M-C. Amoureux et al. I Neuroscience Letters 201 (1995) 61-64
63
MT-III cell line was apparently more resistant to serumdeprivation. Its recovery in serum-supplemented medium was similar to that of plasmid transfected cells although more rapid, since cell survival was less affected during serum-free exposure. These data suggest a protective role for cloned human MT-III in the MT-III transfected CHOKl cell line. Erickson et al. [5] reported stimulation of neuronal survival by MT-III in neuronal cell cultures. Nevertheless, MT-III was originally described as a growth inhibitory factor on neuronal cell survival [22]. It seems that this latter effect can only be measured in the
6
06 0
A 1
2
Exposure
time (days)
3
4
Fig. 2. Comparison of converted MlT between CHO-Kl/pcDNA3 and CHO-Kl/MT-III cells in the absence of serum. Both transfected cell lines were plated at 15 000 cells per well in serum-free conditions. Cell survival is expressed in percentage of the absorbance values obtained at to (0.020-0.039). Curves were constructed using mean values + SD of three independent experiments, each performed in quadruplicate. A, CHO-KUpcDNA3; A, CHO-KlIMT-III. Student’s t-test, **P IO.01 versus CHO-Kl/pcDNA3.
trations of serum (0.05-lo%), and a culture period up to 8 days. A similar cell growth was observed for both cell lines in the presence of 10% foetal calf serum. The exposure time to reach half-maximal converted MTT was obtained between 2.3 and 2.7 days of culture. In contrast, CHO-Kl/pcDNA3 and CHO-Kl/MT-III cells survived differently in the absence of serum. Although cell survival in both cell lines was attenuated by serum deprivation, CHO-KUMT-III cells were more resistant. Converted M?T was 40 + 8% (n = 3; Student’s f-test, PI 0.01) higher in CHO-KUMT-III cells compared to plasmid transfected cells (Fig. 2). Subsequently, cell recovery was measured by supplementing serum to cultures deprived from serum for 24 h. Both cell lines recovered from serum starvation with an almost similar maximal response in converted Ml’T (Fig. 3A). However, a delay (0.87 + 0.12 days; n = 3; Student’s t-test, P IO.01) was observed in the exposure time to reach this effect with the CHO-KUpcDNA3 cell line. A delay was also apparent by measuring [3H]thymidine incorporation; a difference of 1.66 + 0.30 days (n = 3; Student’s f-test, P I 0.05)in the exposure time was measured to attain half-maximal [3H]thymidine incorporation (Fig. 3B). A CHO-Kl cell line was obtained with stable expression of the mRNA for the coding region of human MTIII. The RT-PCR data demonstrated specific expression of human MT-III mRNA; no transcript was found in nonreverse transcribed RNA samples of CHO-KVMT-III cells and reverse transcribed RNA samples of pcDNA3plasmid transfected CHO-Kl cells. The presence of other endogenous MT-proteins in this cell line was not verified and cannot be excluded [19]. The transfected CHO-Kl/
p
0.1
r
E
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0 0
2
4 Exposure
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6 time
a
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(days)
2000
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4
Exposure
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Fig. 3. Recovery of serum-starved CHO-Kl/pcDNA3 and CHOKl/MT-III cells in the presence of serum. Cells were plated at 15 000 cells per well, deprived of serum for 24 h and subsequently exposed to 0.25% foetal calf serum (L). (A) Cell growth was assessed at various times by M’IT conversion. Curves were constructed using mean values rt SD of three independent experiments, each performed in quadruplicate. Exposure times corresponding to half-maximal MTT conversion are 4.2-4.5 days and 5.1-5.7 days for CHO-KlIMT-III and CHOKl/pcDNA3, respectively. (B) Cell growth was quantified by measuring i3H]thymidine incorporation. Curves were constructed using mean values f SD of a representative experiment out of three independent experiments, each performed in quadruplicate. Half-maximal t3H]thymidine incorporation was obtained at 3.7 days and 5.2 days for CHOKUMT-III and CHO-KUpcDNA3, respectively. A, CHO-Kl/pcDNA3; A, CHO-Kl/MT-III.
64
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et (11.I Neuroscience
presence of brain extract [5]. Inhibitory, stimulatory as well as protective effects have now been observed for MT-III on cell survival. These findings argue against the use of the term GIF (growth inhibitory factor) as introduced by Uchida et al. [22] on basis of its growth inhibitory effect. We suggest to use the term MT-III, as it shows high homology with the described MT-proteins
1151. It is plausible that MT-III protects these cells by sharing mechanisms described for other MT-isoforms. The sequence of human MT-III can be aligned with those of other mammalian MT-isoforms so that the position of all 20 constituent cysteine residues is completely conserved. The plethora of cysteine residues and the precision of their arrangement in the polypeptide chain in relation to other MT-isoforms predisposes this molecule to metal binding [7]. Metal detoxification is widely accepted as an important function for MT-proteins [20]. Induction of MT-protein synthesis has been shown to be protective against damage provoked by free radicals, such as by Xirradiation [ll], adriamycin treatment [131, tumour necrosis factor treatment [18], hydroperoxide [14,19] and quinone [3]. Hence, an antioxidant role for MT-proteins may explain the observed protective effects on cell survival [19]. Otherwise, protection by MT-III may be caused by a decreased transcription of functional genes during cell death as suggested by Zeng et al. [23]. In conclusion, a CHO-Kl cell line is available with stable expression of cloned human MT-III that seems to be more resistant to serum-starvation. This cell line will be useful to unravel cell survival effects mediated by MT-III. 111 Amoureux,
M.C., Wurch, T. and Pauwels, P.J., Modulation of metallothionein-III mRNA content and growth rate of rat C6-gliaI cells by transfection with human 5-HTll, receptor genes, Biothem. Biophys. Res. Commun., 214 (1995) 639-645. 121 Braak, H. and Braak, E., Neuropil threads occur in dendrites of tangle bearing nerve cells, Neuropathol. Appl. Neurobiol., 14 (1988) 39-44. [31 Chan, H.M., Tabarrok, R., Tamura, Y. and Cherian, M.G., The relative importance of glutathione and metallothionein on protection of hepatotoxicity of menadione in rat, Chem.-Biol. Interact., 84(1992) 113-124. [41 Crutcher, K.A., Scott, S.A., Lang, S., Everson, W.V. and Weingartner, J., Detection of NGF-like activity in human brain tissue: increased levels in Alzheimer’s disease, J. Neurosci., 13 (1993) 254tX2550. [51 Erickson, J.C., Sewell, A.K., Jensen, L.T., Winge, D.R. and Palmiter, R.D., Enhanced neurotrophic activity in Alzheimer’s disease cortex is not associated with down-regulation of metallothionein-III (GIF), Brain Res., 649 (1994) 297-304. sprouting of cortical neurons PI Ihara, Y., Massive somatodendritic in Alzheimer’s disease, Brain Res., 459 (1988) 138-144.
Letters 201 (1995) 61-64
[71 Kille, P., Hemmings, A. and Lunney, E.A., Memories of metallothionein, Biochim. Biophys. Acta, 1205 (1994) 151-161. Fl Kobayashi, H., Uchida, Y., Ihara, Y., Nakajima, K., Kohsaka, S., Miyatake, T. and Tsuji, S., Molecular cloning of rat growth inhibitory factor cDNA and the expression in the central nervous system, Mol. Brain Res., 19 (1993) 188-194. [91 Lindsay, R.M., Wiegand, S.J., Altar, C.A. and DiStefano, P.S., Neurotrophic factors: from molecule to man, TINS, 17 (1994) 182-190. UOI Manthorpe, M., Fagnani, R., Skaper, SD. and Varon, S., An automated calorimetric microassay for neuronotrophic factors, Dev. Brain Res., 25 (1986) 191-198. [ill Matsubara, J., Shida, T., Ishioka, K., Egawa, S., Inada, T. and Machida, K., Protective effect of zinc against lethality in irradiated mice, Environ. Res., 41 (1986) 558-567. assay for cellular growth and u21 Mosmann, T., Rapid calorimetric survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods, 65 (1983) 55-63. [I31 Nagamuma, A., Satoh, M. and Imura, N., Specific reduction of toxic side effects of adriamycin by induction of metallothionein in mice, Jpn. J. Cancer Res., 79 (1988) 406-411. u41 Ochi, T., Effects of glutathione depletion and induction of metallothioneins on the cytotoxicity of an organic hydroperoxide in cultured mammalian cells, Toxicology, 50 (1988) 257-268. [I51 Palmiter, R.D., Findley, SD., Whitmore, T.E. and Dumam, D.M., MT-III, a brain-specific member of the metallothionein gene family, Proc. Natl. Acad. Sci. USA, 89 (1992) 6333-6337. [I61 Pauwels, P.J., van Assouw, H.P., De Ryck, M., Leysen, J.E., Dom, R. and Van Gool, D., Towards an improved survival of rat brain neurons in culture by cerebrospinal fluid of patients with senile dementia of Alzheimer’s type, Brain Res., 610 (1993) 815. D71 Phillips, H.S., Hains, J.M., Armanini, M., Larami, G.R., Johnson, S.A. and Winslow, J.W., BDNF mRNA is decreased in the hippocampus of individuals with Alzheimer’s disease, Neuron, 7 (1991) 695-702. [181 Sato, M., Sasaki, M. and Honjo, H., Differential induction of metallothionein synthesis by interleukin-6 and tumor necrosis factor-alpha in rat tissues, Int. J. Immunopharmacol., 16 (1994) 187-195. u91 Schwarz, M.A., Lazo, J.S., Yalowich, J.C., Reynolds, I., Kagan, V.E., Tyurin, V., Kim, Y.-M., Watkins SC. and Pitt B.R., Cytoplasmic metallothionein overexpression protects NIH 3T3 from tert-butyl hydroperoxide toxicity, J. Biol. Chem., 269 (1994) 15238-15243. signifi[201 Templeton, D.M. and Cherian, M.G., The toxicological cance of metallothionein, Methods Enzymol., 205 (1991) 11-24. WI Tsuji, S., Kobayashi, H., Uchida, Y., Ihara, Y. and Miyatake, T., Molecular cloning of human growth inhibitory factor and its down-regulation in Alzheimer’s disease, EMBO J., 11 (1992) 4843-4850. WI Uchida, Y., Takio, K., Titani, K., Ihara, Y. and Tomonaga, M., The growth inhibitory factor that is deficient in the Alzheimer’s disease brain is a 68 amino acid metallothionein-like protein, Neuron, 7 (1991) 337-347. [231 Zeng, J., Vallee, B.L. and Kagi, J.H.R., Zinc transfer from transcription factor IBA fingers to thionein clusters, Proc. Natl. Acad. Sci. USA, 88 (1991) 9984-9988.
Neuroscience
titters
201 (1995) 61-64
Expression of human metallothionein-III confers protection against serumfree exposure of stably transfected Chinese hamster ovary CHO-Kl cells Marie-Claude Luborutory
Amoureux, Thierry Wurch, Petrus J. Pauwels*
qf Cellulur and Molecular Neurobiology, Centre de Recherche Pierre Fabre, 17, avenue Jean Moulin, 81106 Castres Cddex, France Received 10 July 199.5;revised version received 16 October 1995; accepted 20 October 1995
Abstract The cloned human metallothionein (MT)-111 coding region tias permanently transfected in Chinese hamster ovary (CHO-Kl) cells in order to investigate the growth regulatory effects of this brain-specific protein. Reverse transcription-polymerase chain reaction (RTPCR) experiments demonstrated stable expression of MT-III mRNA in CHO-KVMT-III cells. Whereas in the presence of serum no differences were observed between the cell growth of both CHO-KUMT-III and CHO-Kl/pcDNA3-plasmid transfected cells, cell survival was attenuated differently in serum-free medium. CHO-Kl/MT-III cells were more resistant (40 + 8%) to serum deprivation than pcDNA3-plasmid transfected cells. Recovery of cell growth for both cell lines was obtained by supplementing serum (0.25%), although with a different
growth rate; half-maximal
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl
tetrazolium
bromide (Mm)-conversion
was ob-
served between 5.1-5.7 and 4.2-4.5 days for CHO-KUpcDNA3 and CHO-Kl/MT-III cells, respectively. These results suggest a protective role for cloned human MT-III, besides its reported inhibitory and stimulatory effects on cell survival. Keywords:
Metallothionein-III (cloned, human); Chinese hamster ovary (CHO-Kl) cell line; Permanent transfection; Reverse transcription-polymerase chain reaction; Cell survival; Serum starvation; Growth factor
Modifications in the brain content of stimulatory (such as nerve growth factor and brain-derived neurotrophic factor) and inhibitory (such as metallothionein (MT)-III) growth factors of Alzheimer’s disease (AD) patients have been reported [4,9,17,22]. MT-III has been shown to be down-regulated in astrocytes of the frontal cortex of AD patients [2 1,221. The authors put forward that reduction in the content of this growth inhibitory factor may stimulate neurotrophic activity of susceptible neurons in the brain by massive sprouting, this latter a well-known feature of AD brain [2,6]. This hypothesis was supported by the observed enhanced neuronal cell survival of rat brain neuronal cultures in the presence of brain extract [5,22] or cerebrospinal fluid [16] of AD patients. Erickson et al. [5], furthermore, showed enhanced neurotrophic activity in frontal cortex of AD patients, not associated, however, with down-regulation of MT-III protein and mRNA. These authors concluded that MT-III-down-regulation is probably not an important pathogenic event in AD. MTIII appeared to be inhibitory on neuronal cell survival in * Corresponding author. Tel.: +33 63 71 42 54; fax: +33 63 71 42 99.
0304-3940/95/$09.50 SSDI
0304-3940(95)
the presence of brain extract. In contrast, neuronal survival was increased when MT-III was added to cultures without brain extract [5]. Therefore, it seems that MT-III is not solely a growth inhibitory factor but may also stimulate neuronal cell survival. The mechanisms involved in regulation of cell survival by MT-III are at present not elucidated. Cloning and sequencing of the human, mouse and rat MT-III genes revealed that it is a member of the MT gene family [8,15,21]. Whereas MT-I and MT-II genes are expressed in many tissues including the brain, MT-III expression is virtually brain-specific [8, 15,221. Even though MT-III is brain-specific, we were interested in the effect of MT-III on the survival and growth of a cell type that did not constitutively express MT-III. Therefore, the coding region of human MT-III was cloned and permanently transfected in Chinese hamster ovary (CHO-Kl) cells. The expression of MT-III mRNA was followed by reverse transcription-polymerase chain reaction (RT-PCR) while MT-III transfected cells were monitored for their survival and growth rate in comparison with a pcDNA3-plasmid transfected CHO-Kl cell line.
0 1995 Elsevier Science Ireland Ltd. All rights reserved 12 134-M
62
M.-C. Amoureux et al. I Neuroscience Letters 201 (1995) 61-64
The human MT-III coding region was amplified from human cerebral cortical cDNA (Clontech, USA) using PCR. A sense primer including a Hind111 restriction site (S-AGTCAAGC’ITATGGACCCTGAGACCTGCCCCTGCCCT-3’) and a reverse primer with a BumHI restriction site (S-AGTCGGATCCTCACTGGCAGCAGCTGCACTTCTCTGC’ITC-3’) were designed to amplify 207 bp of human MT-III cDNA [ 151. The PCR program consisted of 25 repetitive cycles with a denaturing step at 94°C for 1 min, an annealing step at 60°C for 1 min and an extension step at 72°C for 1 min. A single PCR product (200 bp) was inserted in pcDNA3-plasmid (Invitrogen, USA) and sequenced by the dideoxynucleotide chain termination procedure. CHO-Kl cells (lo7 cells, ATCC CCL61) were permanently transfected with 10 pug of pcDNA3/MT-III-plasmid using a gene pulser transfection apparatus (Bio-Rad) at 250 mV and 250pF and grown in nutrient mixture Ham’s F12 supplemented with 10% heat-inactivated foetal calf serum and 1.25 mg/ml geneticin. RNA was extracted using a total RNA separator kit (Clontech, USA), treated with DNaseI (Stratagene, USA) and reverse transcribed into cDNA before PCR was performed as previously described [l]. The sense and reverse primers described above without restriction sites were used to amplify human MT-III cDNA. Control experiments were performed with pcDNA3/MT-III-plasmid, cDNA prepared from human temporal cortical brain tissue, and CHO-Kl cells permanently transfected with pcDNA3-plasmid. The specificity of the amplified PCR products was determined by Ear1 (New England Biolabs, USA) restriction (10 U/PCR reaction for 1 h at 37°C). Quantitative RT-PCR of human MT-III mRNA was compared with the constitutive mRNA /?-actin as previously described [ 11. To follow cell growth and survival of the CHO-Kl cell lines, cells were washed twice with PBS, trypsinised (0.25% trypsin, 15 min at 37”(Z), and plated at the indicated densities either in the absence or presence of serum in 0.25 ml of nutrient mixture Ham’s F12 supplemented with 5pglml transferrin, 30 nM selenium, 5.35 pg/ml linoleic acid, 20 nM progesterone, 1OOpM putrescine, 5 lug/ml insulin and 0.1% bovine serum albumin, in a 24well tissue culture plate. At different times, cell survival was quantified by measuring 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyl tetrazolium bromide (M’IT) conversion as previously described [ 10,121 or measuring [3H]thymidine (1 j&i per well) incorporation. A high yield (90%) of clones of CHO-Kl cells permanently expressing the human MT-III coding region was obtained, as confirmed by RT-PCR; results are shown for one representative clone in Fig. 1A. PCR amplification of 0.2 ng of pcDNA3-plasmid containing the MT-III coding region or 10 ng of RT, DNase-treated human temporal cortical RNA yields a single band of approximately 200 bp (lanes 1, 3). A similar result was obtained with 25 ng of RT, DNase-treated CHO-Kl/MT-III RNA (lane
Fig. 1. Expression of human MT-III mRNA in stably transfected CHOKl/MT-III cells. (A) RNA from CHO-Kl/MT-III cells and post-mortem human brain temporal cortex was isolated, treated with DNaseI and reverse transcribed (RT) before PCR was performed with human MTIII primers. Lane M, 100 bp DNA ladder (Gibco Laboratories, UK); lane 1, 0.2 ng pcDNA3/MT-III; lane 2, Ear1 digestion (10 U) of PCRamplified pcDNA3/MT-III; lane 3, 10 ng RT, DNase-treated human temporal cortical RNA; lane 4, Ear1 digestion of PCR-amplified, RT, DNase-treated human temporal cortical RNA; lane 5, 200 ng DNasetreated human temporal cortical RNA; lane 6,25 ng RT, DNase-treated CHO-KlIMT-III RNA; lane 7, Ear1 digestion of PCR-amplified, RT, DNase-treated CHO-Kl/MT-III cellular RNA; lane 8, 500 ng DNasetreated CHO-KUMT-III RNA; lane 9,500 ng RT, DNase-treated CHOKU pcDNA3 RNA. (B) RNA of CHO-KlIMT-III cells was DNasetreated, reverse transcribed and analysed for MT-III and /I-actin mRNA expression at subcultures 1 (lane l), 5 (lane 2) and IO (lane 3). Quantitative PCR was performed on 4-16 ng, and 0,025-l ng of RT, DNasetreated RNA, with human MT-III and rat B-actin primers, respectively. The figure. corresponds to 8 ng RNA for MT-III and 0.05 ng RNA for B-actin.
6). The enzymatic digestion of these PCR products by Ear1 showed in each case two additional fragments of 119 bp and 88 bp (lanes 2, 4, 7). This further shows that the PCR product amplified from cDNA of the MT-III transfected CHO-Kl cell line is specific for human MTIII. No amplification was measured in samples of DNasetreated human temporal cortical (200 ng) and CHOKl/MT-III (500 ng) RNA without reverse transcriptase treatment (lanes 5, 8) and RT, DNase-treated RNA of CHO-Kl/pcDNA3 cells (500 ng, lane 9). The mRNA expression ratio of MT-III versus /I-actin was stable (between 1.1 and 1.2) for at least ten subcultures (Fig. 1B). The growth of CHO-Kl/pcDNA3 and CHO-Kl/MTIII cells was apparently not different in the presence of serum. This was obvious for various plating densities between 3000 and 150 000 cells per well, various concen-
M-C. Amoureux et al. I Neuroscience Letters 201 (1995) 61-64
63
MT-III cell line was apparently more resistant to serumdeprivation. Its recovery in serum-supplemented medium was similar to that of plasmid transfected cells although more rapid, since cell survival was less affected during serum-free exposure. These data suggest a protective role for cloned human MT-III in the MT-III transfected CHOKl cell line. Erickson et al. [5] reported stimulation of neuronal survival by MT-III in neuronal cell cultures. Nevertheless, MT-III was originally described as a growth inhibitory factor on neuronal cell survival [22]. It seems that this latter effect can only be measured in the
6
06 0
A 1
2
Exposure
time (days)
3
4
Fig. 2. Comparison of converted MlT between CHO-Kl/pcDNA3 and CHO-Kl/MT-III cells in the absence of serum. Both transfected cell lines were plated at 15 000 cells per well in serum-free conditions. Cell survival is expressed in percentage of the absorbance values obtained at to (0.020-0.039). Curves were constructed using mean values + SD of three independent experiments, each performed in quadruplicate. A, CHO-KUpcDNA3; A, CHO-KlIMT-III. Student’s t-test, **P IO.01 versus CHO-Kl/pcDNA3.
trations of serum (0.05-lo%), and a culture period up to 8 days. A similar cell growth was observed for both cell lines in the presence of 10% foetal calf serum. The exposure time to reach half-maximal converted MTT was obtained between 2.3 and 2.7 days of culture. In contrast, CHO-Kl/pcDNA3 and CHO-Kl/MT-III cells survived differently in the absence of serum. Although cell survival in both cell lines was attenuated by serum deprivation, CHO-KUMT-III cells were more resistant. Converted M?T was 40 + 8% (n = 3; Student’s f-test, PI 0.01) higher in CHO-KUMT-III cells compared to plasmid transfected cells (Fig. 2). Subsequently, cell recovery was measured by supplementing serum to cultures deprived from serum for 24 h. Both cell lines recovered from serum starvation with an almost similar maximal response in converted Ml’T (Fig. 3A). However, a delay (0.87 + 0.12 days; n = 3; Student’s t-test, P IO.01) was observed in the exposure time to reach this effect with the CHO-KUpcDNA3 cell line. A delay was also apparent by measuring [3H]thymidine incorporation; a difference of 1.66 + 0.30 days (n = 3; Student’s f-test, P I 0.05)in the exposure time was measured to attain half-maximal [3H]thymidine incorporation (Fig. 3B). A CHO-Kl cell line was obtained with stable expression of the mRNA for the coding region of human MTIII. The RT-PCR data demonstrated specific expression of human MT-III mRNA; no transcript was found in nonreverse transcribed RNA samples of CHO-KVMT-III cells and reverse transcribed RNA samples of pcDNA3plasmid transfected CHO-Kl cells. The presence of other endogenous MT-proteins in this cell line was not verified and cannot be excluded [19]. The transfected CHO-Kl/
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Fig. 3. Recovery of serum-starved CHO-Kl/pcDNA3 and CHOKl/MT-III cells in the presence of serum. Cells were plated at 15 000 cells per well, deprived of serum for 24 h and subsequently exposed to 0.25% foetal calf serum (L). (A) Cell growth was assessed at various times by M’IT conversion. Curves were constructed using mean values rt SD of three independent experiments, each performed in quadruplicate. Exposure times corresponding to half-maximal MTT conversion are 4.2-4.5 days and 5.1-5.7 days for CHO-KlIMT-III and CHOKl/pcDNA3, respectively. (B) Cell growth was quantified by measuring i3H]thymidine incorporation. Curves were constructed using mean values f SD of a representative experiment out of three independent experiments, each performed in quadruplicate. Half-maximal t3H]thymidine incorporation was obtained at 3.7 days and 5.2 days for CHOKUMT-III and CHO-KUpcDNA3, respectively. A, CHO-Kl/pcDNA3; A, CHO-Kl/MT-III.
64
M.-C. Amoureux
et (11.I Neuroscience
presence of brain extract [5]. Inhibitory, stimulatory as well as protective effects have now been observed for MT-III on cell survival. These findings argue against the use of the term GIF (growth inhibitory factor) as introduced by Uchida et al. [22] on basis of its growth inhibitory effect. We suggest to use the term MT-III, as it shows high homology with the described MT-proteins
1151. It is plausible that MT-III protects these cells by sharing mechanisms described for other MT-isoforms. The sequence of human MT-III can be aligned with those of other mammalian MT-isoforms so that the position of all 20 constituent cysteine residues is completely conserved. The plethora of cysteine residues and the precision of their arrangement in the polypeptide chain in relation to other MT-isoforms predisposes this molecule to metal binding [7]. Metal detoxification is widely accepted as an important function for MT-proteins [20]. Induction of MT-protein synthesis has been shown to be protective against damage provoked by free radicals, such as by Xirradiation [ll], adriamycin treatment [131, tumour necrosis factor treatment [18], hydroperoxide [14,19] and quinone [3]. Hence, an antioxidant role for MT-proteins may explain the observed protective effects on cell survival [19]. Otherwise, protection by MT-III may be caused by a decreased transcription of functional genes during cell death as suggested by Zeng et al. [23]. In conclusion, a CHO-Kl cell line is available with stable expression of cloned human MT-III that seems to be more resistant to serum-starvation. This cell line will be useful to unravel cell survival effects mediated by MT-III. 111 Amoureux,
M.C., Wurch, T. and Pauwels, P.J., Modulation of metallothionein-III mRNA content and growth rate of rat C6-gliaI cells by transfection with human 5-HTll, receptor genes, Biothem. Biophys. Res. Commun., 214 (1995) 639-645. 121 Braak, H. and Braak, E., Neuropil threads occur in dendrites of tangle bearing nerve cells, Neuropathol. Appl. Neurobiol., 14 (1988) 39-44. [31 Chan, H.M., Tabarrok, R., Tamura, Y. and Cherian, M.G., The relative importance of glutathione and metallothionein on protection of hepatotoxicity of menadione in rat, Chem.-Biol. Interact., 84(1992) 113-124. [41 Crutcher, K.A., Scott, S.A., Lang, S., Everson, W.V. and Weingartner, J., Detection of NGF-like activity in human brain tissue: increased levels in Alzheimer’s disease, J. Neurosci., 13 (1993) 254tX2550. [51 Erickson, J.C., Sewell, A.K., Jensen, L.T., Winge, D.R. and Palmiter, R.D., Enhanced neurotrophic activity in Alzheimer’s disease cortex is not associated with down-regulation of metallothionein-III (GIF), Brain Res., 649 (1994) 297-304. sprouting of cortical neurons PI Ihara, Y., Massive somatodendritic in Alzheimer’s disease, Brain Res., 459 (1988) 138-144.
Letters 201 (1995) 61-64
[71 Kille, P., Hemmings, A. and Lunney, E.A., Memories of metallothionein, Biochim. Biophys. Acta, 1205 (1994) 151-161. Fl Kobayashi, H., Uchida, Y., Ihara, Y., Nakajima, K., Kohsaka, S., Miyatake, T. and Tsuji, S., Molecular cloning of rat growth inhibitory factor cDNA and the expression in the central nervous system, Mol. Brain Res., 19 (1993) 188-194. [91 Lindsay, R.M., Wiegand, S.J., Altar, C.A. and DiStefano, P.S., Neurotrophic factors: from molecule to man, TINS, 17 (1994) 182-190. UOI Manthorpe, M., Fagnani, R., Skaper, SD. and Varon, S., An automated calorimetric microassay for neuronotrophic factors, Dev. Brain Res., 25 (1986) 191-198. [ill Matsubara, J., Shida, T., Ishioka, K., Egawa, S., Inada, T. and Machida, K., Protective effect of zinc against lethality in irradiated mice, Environ. Res., 41 (1986) 558-567. assay for cellular growth and u21 Mosmann, T., Rapid calorimetric survival: application to proliferation and cytotoxicity assays, J. Immunol. Methods, 65 (1983) 55-63. [I31 Nagamuma, A., Satoh, M. and Imura, N., Specific reduction of toxic side effects of adriamycin by induction of metallothionein in mice, Jpn. J. Cancer Res., 79 (1988) 406-411. u41 Ochi, T., Effects of glutathione depletion and induction of metallothioneins on the cytotoxicity of an organic hydroperoxide in cultured mammalian cells, Toxicology, 50 (1988) 257-268. [I51 Palmiter, R.D., Findley, SD., Whitmore, T.E. and Dumam, D.M., MT-III, a brain-specific member of the metallothionein gene family, Proc. Natl. Acad. Sci. USA, 89 (1992) 6333-6337. [I61 Pauwels, P.J., van Assouw, H.P., De Ryck, M., Leysen, J.E., Dom, R. and Van Gool, D., Towards an improved survival of rat brain neurons in culture by cerebrospinal fluid of patients with senile dementia of Alzheimer’s type, Brain Res., 610 (1993) 815. D71 Phillips, H.S., Hains, J.M., Armanini, M., Larami, G.R., Johnson, S.A. and Winslow, J.W., BDNF mRNA is decreased in the hippocampus of individuals with Alzheimer’s disease, Neuron, 7 (1991) 695-702. [181 Sato, M., Sasaki, M. and Honjo, H., Differential induction of metallothionein synthesis by interleukin-6 and tumor necrosis factor-alpha in rat tissues, Int. J. Immunopharmacol., 16 (1994) 187-195. u91 Schwarz, M.A., Lazo, J.S., Yalowich, J.C., Reynolds, I., Kagan, V.E., Tyurin, V., Kim, Y.-M., Watkins SC. and Pitt B.R., Cytoplasmic metallothionein overexpression protects NIH 3T3 from tert-butyl hydroperoxide toxicity, J. Biol. Chem., 269 (1994) 15238-15243. signifi[201 Templeton, D.M. and Cherian, M.G., The toxicological cance of metallothionein, Methods Enzymol., 205 (1991) 11-24. WI Tsuji, S., Kobayashi, H., Uchida, Y., Ihara, Y. and Miyatake, T., Molecular cloning of human growth inhibitory factor and its down-regulation in Alzheimer’s disease, EMBO J., 11 (1992) 4843-4850. WI Uchida, Y., Takio, K., Titani, K., Ihara, Y. and Tomonaga, M., The growth inhibitory factor that is deficient in the Alzheimer’s disease brain is a 68 amino acid metallothionein-like protein, Neuron, 7 (1991) 337-347. [231 Zeng, J., Vallee, B.L. and Kagi, J.H.R., Zinc transfer from transcription factor IBA fingers to thionein clusters, Proc. Natl. Acad. Sci. USA, 88 (1991) 9984-9988.