Transformation and characterization of mutant human fibroblasts defective in peroxisome assembly

EXPERIMENTAL

CELL

RESEARCH

201,307-312

(19%)

Transformation and Characterization of Mutant Human Fibroblasts Defective in Peroxisome Assembly H. OKAMOTO, Department

Y. SUZUKI,~ of Pediatrics,

School

N. SHIMOZAWA, of Medicine,

Gifu

Inc.

INTRODUCTION

In cultured skin fibroblasts from patients with Zellweger’s cerebrohepatorenal syndrome (ZS), neonatal adrenoleukodystrophy (NALD), and infantile Refsum disease there is an abnormality in the assembly of peroxisomes [l]. Patients with these autosomal recessive diseases have severe psychomotor retardation, muscle weakness, liver dysfunction, and a dysmorphic appearance. Death usually occurs in early infancy. Multiple enzyme defects in these patients including absence of P-oxidation enzymes [2], enzymes in the biosynthesis of plasmalogen [3], phytanic acid oxidase [4], pipecolic acid oxidase [5], and nonspecific lipid transfer protein 1 To whom

reprint

requests

should

University,

M. MASUNO,

Tsukasa-machi

AND T. ORII

40, Gifu

500, Japan

[6] and aberrant intracellular distribution of catalase [7] are considered to be caused by defects in peroxisome biogenesis. The mechanisms of peroxisome biogenesis and the primary etiology of these inherited diseases are not well understood. Cultured skin fibroblasts have been used for molecular and cytochemical analyses of these disorders; however, the limited proliferative capacity combined with a limited speed of growth and cell population present problems when one attempts to clone a gene necessary for the biogenesis of peroxisomes. Utilization of mutant animal cell lines with a more rapid growth is one approach that can be used to overcome these problems. Mutant Chinese hamster ovary cells with a defect in the assembly of peroxisomes have been isolated [8], and a complementing gene (peroxisome assembly factor-l) for one of the mutant cell lines was cloned [9]. If genetic relationships between these mammalian cell models and human diseases are clarified, results in the mammalian cells could be applied to human diseases. We have recently clarified a primary gene lesion in a patient with ZS who belonged to the same complementation group as that of a mammalian cell line with defective peroxisome assembly factor-l ]lO]. Establishment of transformed human fibroblast cell lines with deficient peroxisomes is another feasible approach. At least eight genetic forms of peroxisome-deficient disorders were identified in a complementation study; the correction of peroxisome biogenesis tested by the immunofluorescence staining of peroxisomes was examined [ 111. It seemslikely that numerous genes necessary for peroxisome biogenesis can be cloned by complementing gene analyses of these human cell lines. We obtained transformed fibroblasts from a patient with ZS and one with NALD; they belong to different genetic complementation groups. These transformants exhibit a higher proliferative capacity and maintain the biochemical abnormalities and genetic properties of the parental cells.

Human skin fibroblasts deficient in peroxisome biogenesis were transformed by transfecting SV40 orii DNA with the use of an electroporator, and the biochemical, immunocytochemical, and cytogenetic properties of the transformants were analyzed. Cells (1 X 10’) from a patient with Zellweger syndrome and one with neonatal adrenoleukodystrophy were suspended with 2 wg of SV40 orii DNA in PBS; then a high-voltage pulse (2000 V, 30 ~.ls) was generated two times. Several colonies expressing large T-antigen were picked up 4 weeks after transfection. Doubling time of the transformants was about half of that and the saturation density was 5 to 10 times greater than that of the parental cells. Biochemical abnormalities including defective lignoceric acid oxidation, dihydroxyacetone phosphate acyltransferase deficiency, and disturbed biosynthesis of peroxisomal B-oxidation enzymes were preserved in the transformants. Peroxisomes were defective in all colonies, as determined by immunofluorescence staining using anti-catalase IgG. Cell fusion studies confirmed that the transformants belong to the same complementation groups as those of the parental cells. These transformed mutant cell lines are expected to be useful tools for investigating the pathogenesis of inherited diseases related to defects in peroxisome biogenesis. o ISSZ Academic Press,

S. YAJIMA,

MATERIALS

AND

Cell culture and transformation. subject, a patient with ZS (TI),

be addressed.

METHODS

Skin fibroblasts and one with NALD

307 All

Copyright Q 1992 rights of reproduction

from

a control

(RS) were cul-

0014.4827/92 $5.00 by Academic Press, Inc. in any form reserved.

308

OKAMOTO

FIG. 1. lmmunocytochemical staining of large T-antigen. Cells were fixed with methanol and stained with anti-T-Ag IgG and alkaline phosphatase-conjugated anti-mouse IgG. In the transformed SVRSa cells (B), nuclei were strongly stained. (A) Untransformed fibroblasts. Bar = 100 pm.

tured in Eagle’s minimum essential medium (MEM) supplemented with 10% fetal calf serum (FCS). Cell lines Tl and RS belong to the complementation groups B and E, respectively [ll]. The passage number of these fibroblast cell lines was between six and eight when we used them for transformation. Clinical and biochemical findings for these patients have been described elsewhere [12, 131. About 1 X 10s cells were trypsinized with 0.1% trypsin/phosphatebuffered saline (PBS), washed twice with PBS, and suspended in 0.25 ml of PBS. Two micrograms of SV40 ori- DNA, obtained from the Cancer Research Resources Bank of Tokyo, Japan, was cut with the restriction enzyme Xhol, added to the preparation, and kept on ice for 10 min. Transfection of SV40 DNA into fibroblasts was performed by electroporation [14] using electroporator ESCF-3001 (ESCO Limited, Tokyo, Japan). Cell suspension was introduced into a disposable cell chamber ECB-1004 (Rikoh Kagaku Lab., Inc., Tokyo, Japan) and a high-voltage electric pulse (5000 V/cm, 30 ps) was generated two times. After electroporation, the cell suspension was kept at room temperature for 5 min and the cells were seeded into a 50-cm2 dish. MEM supplemented with 10% FCS was replaced every 3 days. About 4 weeks after this transfection, colonies of the transformants had grown to a diameter of 1 mm; each colony was picked up using a micropipet. The cells were dispersed and cultured in small dishes. Transformation was confirmed by morphologic changes, proliferative capacity, and expression of large T-antigen, detected by the immunoblot analysis [15] and immunocytochemical staining [16], using mouse monoclonal anti-T-Ag IgG (Oncogene Science, Inc., Manhasset, NY) and alkaline phosphatase-conjugated anti-mouse IgG (Promega, Madison, WI). Biochemical and somatic cell genetic properties of the transformants. Activity of the peroxisomal P-oxidation was assessed by

ET

AL.

lignoceric acid oxidation [ 171. Confluent cells in an &cm2 dish were preincubated with FCS-free MEM for 1 h and then incubated with 4 nmol (2 X 10 cpm) of [l-‘4C]lignoceric acid (46.7 mCi/mmol; CEA, Gif-Sur-Yvette, France) in FCS-free MEM for 1 h. Radiolabeled acidsoluble degradation products in the medium were extracted and counted. Activity of dihydroxyacetone phosphate (DHAP) acyltransferase was assayed as described before [16]. [U-i4C!]DHAP was generated from [U-i4C]glycerol 3-phosphate (Amersham, Buckinghamshire, UK). Radioactivity incorporated into the chloroform-soluble lipid was determined. Biosynthesis of peroxisomal ,&oxidation enzymes and catalase was analyzed in pulse-chase experiments [18]. Cells in three dishes (8 cm*) were preincubated with methionine-free MEM supplemented with 5% dialyzed FCS for 1 h and then pulse-labeled with 0.2 mCi of [35S]methionine (>lOOO Ci/mmol; Amersham, Buckinghamshire, UK) in the same medium as that used at the preincubation. The cells were either harvested immediately after pulse labeling or were chased with MEM supplemented with 10% FCS and methionine for 6 or 24 h and then harvested. The cell lysate was immunoprecipitated with antibodies against acyl-CoA oxidase, 3-ketoacyl-CoA thiolase, and catalase, a kind gift from Professor T. Hashimoto (Shinshu University School of Medicine, Matsumoto, Japan). lmmunoprecipitates were subjected to sodium dodecyl sulfate/polyacrylamide gel electrophoresis and fluorography. lmmunofluorescence staining was performed as described [6]. Cells were cultured on cover slips, fixed with 4% paraformaldehyde/0.15 M potassium phosphate buffer, pH 7.4, for 1 h, and permeabilized with buffer A (0.1% Triton X-loo/PBS) for 15 min. After blocking with 2% FCS/buffer A, the cells were incubated with anti-human catalse for 2 h, washed three times with PBS, and then reincubated with fluorescein isothiocyanate-conjugated goat F(ab’)2 anti-rabbit IgG (TAGO, Burlingame, CA) for 1 h. Cover slips were washed with PBS, mounted on slide glass, and observed under a fluorescence microscope (Nikon VSF-R, Tokyo, Japan). Complementation study of transformants and parental fibroblasts was as described elsewhere [ 111. A mixture of two cell lines was cocultivated for 1 day. The cells were fused in the presence of 45% polyethylene glycol for 1 min, washed four times with FCS-free MEM, and then cultured in MEM supplemented with 10% FCS. Three days after fusion, the fused cells were stained as described above, and the formation of peroxisomes was observed.

AND

RESULTS

Transformation

of Diploid

DISCUSSION

Fibroblasts

All the cell lines used were readily transfected by electroporation. Ten to twenty colonies of transformants

-9OkD

12

3

4

5

FIG. 2. lmmunoblot analysis were subjected to SDS/polyacrylamide noblot analysis using anti-T-Ag IgG gated second antibody. In lanes 1,2 @V-TIC), 6 (SV-RSa), 7 (SV-RSh), with the molecular weight of 90 kDa 5, untransformed cell lines.

6

7

8

of large T-antigen. Cell extracts gel electrophoresis and immuand alkaline phosphatase-conju(control transformed cell lines), 4 and 8 (SV-RSc), large T-antigen was clearly detected. Lanes 3 and

TRANSFORMATION

OF

PEROXISOME-DEFICIENT

TABLE Characterization Doubling time (h) 52 70 64 24 23 30 32

Control TI (ZS) RS (NALD) SV-TIa (ZS) SV-TIC (ZS) SV-RSa (NALD) SV-RSc (NALD) Note. done.

Saturation density (cells/cm’)

DHAP-AT,

3.3 2.4 3.0 24.0 18.4 16.0 13.6

dihydroxyacetone

x X x X X x x

1

of Parental and Transformed

Lignocerate oxidation (pmol/h mg protein) lo4 lo4 lo4 lo4 lo4 lo4 lo4 acyltransferase;

Fibroblasts

DHAP-AT activity (nmol/h mg protein)

391 7 53 0 0 61 48

phosphate

1.00 0.07 0.09 nd 0.08 nd 0.16 ZS, Zellweger

were grown from 1 X lo6 fibroblasts 4 weeks after the transfection. Three colonies from ZS (SV-TIa, SV-TIb, SV-TIC) and NALD (SV-RSa, SV-RSb, SV-RSc) were cultivated. Figure 1B shows expression of the large Tantigen in the nuclei of the transformants. In the parental fibroblasts, large T-antigen was not detected (Fig. 1A). A faint staining in the untransformed cells was nonspecific due to a long exposure in color developing solution. Large T-antigen was also detected by immunoblot analysis. Ninety kilodaltons of large T-antigen was evident in all the transformed cell lines (Fig. 2). Doubling time of transformants was from 23 to 32 h; that is, about half that of the parental fibroblasts. Satu-

309

FIBROBLASTS

syndrome;

NALD,

Complementation Peroxisomes

group WI

Present Absent Decreased Absent Absent Decreased Decreased

B E B B E E

neonatal

adrenoleukodystrophy;

nd, not

ration density when the cells become confluent was from 1.4 X lo5 to 2.4 X lo5 cells/cm’, that is, about 5 to 10 times that of the untransformed cells (Table 1). Figure 3 shows the growth curve when 2 X lo5 cells were seeded in a 25cm2 dish. Transformants rapidly grew to 5 X lo6 cells 1 week after seeding. However, the trans-

number of cells

O, a: transformed ., A: parental

cell cell

123456789 5

10

FIG. 3. Growth curve of transformed and lines: 2 X lo5 cells per dish (25 cm*) were seeded Doubling time of transformants was about half of tion density was 5 to 10 times greater than that lines. 0, TI; A, RS; 0, SV-TIa; a, SV-RSa.

15 days

untransformed cell and counted daily. that and the saturaof the parental cell

FIG. 4. Pulse-chase experiments in transformed cell lines. Cells were pulse-labeled with [?S]methionine and chased for 6 or 24 h. The cell lysate was immunoprecipitated with each antibody and subjected to SDS/polyacrylamide gel electrophoresis and fluorography. (A) Acyl-CoA oxidase; (B) 3-ketoacyl-CoA thiolase; (C) catalase. Lanes 1, 2, and 3, control-transformed cell line (pulse, chase 6 h, chase 24 h); lanes 4,5, and 6, SV-TIC (pulse, chase 6 h, chase 24 h); lanes 7,8, and 9, SV-RSc (pulse, chase 6 h, chase 24 h).

310

OKAMOTO

ET

AL.

under way. These transformants continued to grow after the 100 population doubling level and chromosome analysis revealed that these transformants showed an unstable pseudotetraploid karyotype. Biochemical and Immunocytochemical Transformants

Properties

of the

Peroxisomal P-oxidation activity assessed by the lignoceric acid oxidation was deficient in both parental fibroblasts and transformants (Table 1). The severity of the defect in the transformants correlated well with that in the parental cells [ 171. Deficient activity of DHAP acyltransferase was also maintained in the transformants. Biosynthesis of peroxisomal enzymes, as analyzed by the pulse-chase experiments, was also disturbed in the transformants, in the same manner as that seen in the parental cells [18]. Acyl-CoA oxidase, a peroxisomal poxidation enzyme, was first synthesized as a 75kDa polypeptide and then was processed to 53- and 22-kDa polypeptides in the control transformants (Fig. 4A, lanes 1,2, and 3). In the transformants SV-TIa and SV-

FIG. 5. Immunofluorescence staining of peroxisomes. Procedures are described under Materials and Methods. (A) Control untransformed fibroblasts; (B) parental fibroblasts from patient RS (NALD); (C) parental fibroblasts from patient TI (ZS); (D) control transformed fibroblasts; (E) transformant SV-RSa; (F) transformant SV-TIb. Bar = 10 Km.

formants could not be maintained at a confluent state, and early reseeding was necessary. The modal numbers of chromosomes in the transformants W-TIC and SVRSc were 87 (range, 84-90) and 82 (range, 79-85), respectively. SV40 orii DNA is useful for transformation of human cells [ 193, although immortalization rarely occurs. Previous methods used for transformation including calcium phosphate coprecipitation [ZO] are laborious and inefficient. The present method by electroporation is simple, there is no need for special reagents, and a number of colonies of transformants can be acquired. The basic conditions of electroporation were examined and we found voltage to be the most important factor. However, the efficacy of transformation differed among the cell lines used [14]. Evaluation of immortalization is

FIG. 6. Immunofluorescence fused cells. Experimental procedures and Methods. In A (RS X SV-RSa) no peroxisomes was formed. In B RSa), abundant peroxisomes were

staining of peroxisomes in the are described under Materials and C (TI x SV-TIb), practically (RS X SV-TIC) and D (TI X SVvisualized. Bar = 10 @cm.

TRANSFORMATION

SVmTla I SVmTlb SV-Tic

TI

RS

OF

SV-RSa

SV-RSb

PEROXISOME-DEFICIENT

SV RSc

/ TI

/

SV-TIC

US SV-RSa

~

1 -:----.--~I

+

-,

f

\I

SVmTla SVmTlb ~~~~

-

-$*,~ 1 1 ~~~ \ ) ‘+ I \

+ mm~_m~‘p

~~~ Lmm

-+-+

K-r ’ t /

---;-

.~

-1

SV RSb SV RSc

._~ +

; I

i

I I,

i 1

>y--

-~---

~ ~~

~~ _cIl--

1~

i

I

I

FIG. 7. Complementation study of transformed and parental cell lines. TI and its transformants (SV-Tla, SV-Tlb, SV-TIC) and RS and its transformants (SV-RSa, SV-RSb, SV-RSc) belong to the same complementation groups, respectively. +, complemented; -, not complemented.

RSa, this processing was greatly disturbed (lanes 4 to 9). Only a small amount of 75-kDa polypeptide was processed to the 53-kDa polypeptide in SV-RSa. 3-Ketoacyl-CoA thiolase, another peroxisomal P-oxidation enzyme, was synthesized as a 43-kDa precursor in all transformants. However, the processing to the 41-kDa mature protein was disturbed in SV-TIa and SV-RSa (Fig. 4B). Biosynthesis of catalase was not disturbed in any transformant as was the case for the untransformed fibroblasts (Fig. 4C). Immunofluorescence staining revealed that transformants maintained the properties of peroxisomes in the parental fibroblasts. Peroxisomes were abundant in the control transformants (Fig. 5D). In the transformants SV-RSa, peroxisomes were detected in about 10% of the cells as was the case for the parental cells (Figs. 5B and 5E). Few or no peroxisomes were detected in the ZS fibroblasts and transformants SV-TIb (Figs. 5C and 5F). These results indicate that the transformants maintained the original biochemical and immunocytochemical properties of the parental cell lines. Furuya et al. [21] and our group [ 141 found that several transformed fibroblasts from patients with lysosomal storage diseases carried the same enzyme defects as those of parental cell lines. Confirmation of the original biochemical defect is essential since the integration of SV40 DNA occurs at random locations.

311

FIBROBLASTS

These results indicate that the parental cell lines and their transformed cell lines belong to the same complementation groups and that genetic defects of the parental cell lines are apparently maintained in the transformed cell lines. On the other hand, peroxisomes were abundantly formed in fused cells, using RS and SV-TIC (Fig. 6B), TI and SV-RSa (Fig. 6D), TI and RS, or SVTIC and SV-RSc (Fig. 7). Thus, the genetic heterogeneity in the parental fibroblasts was considered to be maintained in these transformants. These somatic cell genetic studies suggest that the transformed fibroblasts can be used for molecular analysis of the primary etiology of absence of peroxisomes. We obtained evidence for the same point mutation in the P-glucuronidase gene in the transformed fibroblasts from a patient with mucopolysaccharidosis type VII [ 141. As we found that at least eight complementation groups were present [ll], numerous factors (genes) are considered to be involved in the formation of peroxisomes or in the translocation of peroxisomal proteins. Studies of the primary etiology of each complementation group using transformed fibroblasts will pave the way toward elucidation of the entire structure of peroxisome biogenesis. We are grateful to Professor T. Hashimoto for the gift of antibodies and to M. Ohara for helpful comments. This study was supported in part by Grant-in-Aid for Scientific Research 03770558 from the Ministry of Education, Science and Culture of Japan, a research grant for study of intractable diseases from the Ministry of Health and Welfare of Japan, and a grant from Uehara Memorial Life Science Foundation.

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Peroxisomes did not form in fused cells when RS and SV-RSa (Fig. 6A), TI and SV-TIb (Fig. 6C), SV-TIa and SV-TIb, or SV-RSa and SV-RSb were used (Fig. 7).

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