Hum Genet (1989) 81:165-170

© Springer-Verlag 1989

Ataxia-telangiectasia fibroblasts have less fibronectin m R N A than control cells but have the same levels of integrin and [3-actin m R N A

Yechiel Becker, Eynat Tabor, and Yael Asher

Department of Molecular Virology, Faculty of Medicine, Hebrew University of Jerusalem, Jerusalem, Israel

Summary. The expression of fibronectin, integrin and ~-actin genes in skin fibroblasts from patients with the genetic dis- order ataxia-telangiectasia (A-T) was studied. These three genes were selected because their protein products contribute to the shape and function of the fibroblast. Expression of mRNA by these genes was compared with that in fibroblasts from normal individuals and from patients with the genetic disorder, hereditary hemorrhagic telangiectasia (HHT). A-T fibroblasts were found to produce less fibronectin mRNA than normal and HHT fibroblasts. A-T fibroblasts senesce around passage level 15 while normal and HHT fibroblasts can be propagated for many more passages in vitro. However, the expression of the integrin gene in A-T fibroblasts was simi- lar to that in normal fibroblasts, while the ~3-actin gene was ex- pressed at a higher level. The increased 13-actin mRNA levels were similar in fibroblasts of patients with the two genetic dis- orders, A-T and HHT, but higher than in normal fibroblasts. HHT fibroblasts differed markedly from A-T fibroblasts in having a high level of fibronectin and integrin mRNA expres- sion. The results indicate that regulation of the fibronectin gene in A-T fibroblasts differs from that of the integrin and [3- actin genes, and that the decline in fibronectin m R N A may be linked to the shortened in vitro life-span of these cells.

Introduction

A-T is an autosomal recessive human genetic disorder, the manifestations of which in homozygous children are pleio- tropic and include progressive cutaneous telangiectasias, progeric hair and skin changes, growth retardation, endocrine abnormalities (including ovarian dysgenesis and insulin-resis- tant diabetes), hypoplasia or absence of the thymus gland, ab- normal immune mechanisms and immune deficiency, ataxia and degenerating Purkinje cells in the brain, proneness to lymphoreticular neoplasia, clinical and cellular radiosensitiv- ity, chromosomal instability and D N A repair defect (Boder 1985). Numerous studies have been published on clinical and molecular aspects of A-T, many of which have also been sum- marized and discussed in conferences (see, for example, Bridges and Harnden 1982; Gatti and Swift 1985). In spite of the research efforts to identify the A-T recessive gene, it is not yet known which human gene is involved in the genetic defect and is responsible for the pleotropic effects seen in many or- gans of homozygous patients.

Offprint requests to: Y. Becker

A-T fibroblasts have a short life-span in culture and an altered cellular morphology; they respond to epidermal growth factor (EGF) and to fibroblast growth factor (FGF) (Shiloh et al. 1982). Various studies (reviewed in Becker 1986) revealed that actin fibers are detached from their an- chorage in A-T fibroblasts. It was also reported that the medium of A-T fibroblasts contains higher amounts of fibro- nectin compared to normal fibroblasts (Murnane and Painter 1983). A similar observation on overproduction of fibronectin was made by M. Tatsuka (personal communication), using fibroblasts from Japanese A-T patients. Murnane and Painter (1983) used antiserum to plasma fibronectin to detect fibro- nectin in A-T fibroblasts and in the culture medium. The fibronectin secreted from the A-T cells was 10kDa smaller than the fibronectin released into the medium by normal fibroblasts. Lavin and Seymour (1984) have reported on reduced levels of fibronectin in A-T lymphoblastoid cells.

The above findings, together with the observation of de- tached actin fibers in A-T fibroblasts (see Becker 1986) sug- gested a possible disturbance in the organization of the A-T fi- broblasts. Since cellular fibronectin molecules bind extracellu- larly to the integrin proteins situated in the outer cell membrane (Hynes and Yamada 1982), to which actin fibers bind intracel- lularly, a disturbance in one of these proteins might lead to ab- normal behavior of the A-T fibroblast and hence to the biologi- cal phenomena described above. Thus, we decided to study the expression of the genes coding for fibronectin (plasma and cellular forms), integrin and J3-actin in A-T fibroblasts.

Studies on the expression of fibronectin genes were made possible by cloning the rat and human fibronectin genes and by identification of the human genes for plasma and cellular fibronectin. The three genes for fibronectin were identified in the following human chromosomes: chromosome 2; p14-p16; chromosome 2, q34-q36; chromosome 11, q12.1-q13.5 (Ya- mada 1983). One of the genes in chromosome 2 is thought to be the gene for plasma fibronectin and the second is a pseudo- gene. The fibronectin gene in chromosome 11 is thought to code for the cellular fibronectin (Jahnwar et al. 1986; Prowse et al. 1986). The gene for rat fibronectin was found to contain 48 exons, and is thus one of the most complex genes known (Schwarzbauer et al. 1983; Kornblihtt et al. 1983).

Colombi et al. (1986) reported that normal human fibro- blast lines (Flow 7000 and HEL 299) produce three fibronec- tin mRNA species, namely two of 8.6 and 8.0 kb, respectively, which are ED ÷ and one mRNA species of 7.Tkb which was ED- . The ED ÷ mRNA species contains a 270bp insert desig- nated "extra domain" (ED+), which corresponds to one exon in the human fibronectin gene (Vibe-Pedersen et al. 1984) and

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encodes one of the homologous type III repeats of the protein which seems to be absent in plasma fibronectin (Kornblihtt et al. 1983). The presence of spliced heterogenous fibronectin mRNA species in human fibroblasts indicates that alternative splicing occurs in cultured human cells, leading to release of both the plasma form of fibronectin lacking the ED sequence and cellular fibronectin which retains it (Kornblihtt et al. 1983). Since human cells were found to have two genes for fibronectin (Yamada 1983), it is possible that splicing of fibro- nection mRNA species from the two genes is responsible for the synthesis of both cellular and plasma fibronectin species.

The fibronectin in the fibrils of the extracellular matrix align with intracellular microfilament bundles containing actin (Hynes and Destree 1978; Hynes and Yamada 1982). The pres- ence of a physical connection, in the form of glycoproteins, between the extracellular fibronectin and intracellular actin fibers was postulated by Hynes (1981) to play a significant role in cellular adhesion, cellular morphology and migration. The 140 kDa group of glycoproteins (designated "integrin") con- sists of three distinct glycoproteins in chicken fibroblasts and of two glycoproteins in mammalian cells (Tamkun et al. 1986). A gene which codes for one of the three 140kDa glycoproteins in the chicken cells was cloned and found to code for 3.72kb mRNA and the protein integrin of 803 amino acids. Although the human gene coding for integrin glycoproteins is not known, the integrin DNA probe can be used to identify inte- grin mRNA in human fibroblasts.

The human gene which codes for the cytoplasmic actin fibers (the [3-actin gene) resides in chromosome 7 p t e r - q l l (Nudel et al. 1983). This gene codes for an mRNA species of 1.6 kb and can be detected by a probe derived from the cloned gene. In the present study, we analyzed by the Northern hy- bridization technique the contents of mRNA transcribed from the fibronectin, integrin and 13-actin genes in A-T fibroblasts cultured in vitro. In addition we used skin fibroblasts from a normal individual as a control. As an additional control, two fibroblast lines from two patients with the genetic disorder hereditary hemorrhagic telangiectasia (HHT), also known as Rendu-Osler-Weber syndrome (Hanes 1909) were studied. Inheritance of this genetic disorder is presumed to be auto- somal dominant, with the late appearance of typical lesions (Garland and Anning 1950).

HHT usually occurs in childhood, most commonly in Jews and Anglo-Saxons, and less frequently, in Blacks (Posner and Sampliner 1978; Guena and Addei 1982). The syndrome is characterized by multiple small telangiectasic lesions of skin, mucous membranes and viscera, a tendency of the lesions to bleed, and a relative frequency of pulmonary arterio-venous fistulas. These are followed by hemoptysis and neurological symptoms (Stanley and Hunter 1970). As a result of chronic blood loss, patients develop iron deficiency anemia, and occa- sionally life-threatening hemorrhages occur (Guena and Addei 1982). The development of telangiectasias and neurological disorders in HHT patients resembles the development of telangiectasias and ataxia in patients with A-T, also known as the Louis-Bar syndrome (Louis-Bar 1941). For this reason, the properties of HHT were investigated and compared to those of A-T.

The present study revealed that the level of mRNA trans- cripts from fibronectin genes was markedly reduced in A-T fibroblasts when a late passage was reached in vitro, while normal mRNA levels were found with the integrin and actin gene probes. HHT fibroblasts differed markedly from A-T

fibroblasts in having increased levels of integrin and actin mRNAs and unchanged fibronectin mRNA levels compared to normal fibroblasts.

Materials and methods

Cel/s

The fibroblast strains used in this study were: GM 3418, GM 3419 - skin fibroblasts obtained from two HHT patients, GM 5823 - skin fibroblasts from an A-T patient, and GM 0037D - normal fibroblasts. These cell cultures were obtained from the Coriell Institute for Medical Research, Camden N.J. The cells were propagated and samples were frozen in liquid air. F-193 skin fibroblasts were obtained from a normal donor and F-28 skin fibroblasts were obtained from an A-T patient (Jewish-Moroccan origin) in Jerusalem, by the Human Ge- netics Laboratory, Hadassah Hospital, Hebrew University- Hadassah Medical Center, Jerusalem. Two additional A-T fibroblast cultures, F-320 and F-59 were also studied. The A-T fibroblast cultures were frozen in liquid nitrogen at passage levels 4-5 and were thawed, cultured and studied without fur- ther freezing. Chick primary fibroblasts were prepared from specific-pathogen-free (SPF) eggs.

Cells were propagated in Dulbecco's modified Eagle's medium (Grand Island Biologicals, Grand Island, N.Y.), supplemented with 15% (v/v) fetal calf serum (GIBCO) and were free of Mycoplasma. Since A-T fibroblasts have a limited life-span (about 15-20 passages at most) in culture (Shiloh et al. 1982), we used the fibroblasts at a stage when their growth was still active. Total cellular RNA was extracted from the cells at passage levels 8-9 and 13-15, after reaching confiuency at 3 days of cultivation.

Preparation of cellular RNA

Total cellular RNA was isolated by the guanidinium-isothio- cyanate method (Chirgwin et al. 1979). The extracted RNA was dissolved in water and the RNA content of each prepara- tion was determined. Samples containing 5 gg of RNA were loaded per slot and electrophoresed in 1.5% agarose gels. The RNA content of ribosomal RNA was checked prior to and after transfer to nitrocellulose filters (Schleicher and Schuell, Dassel, FRG) by the Northern blot technique (Thomas 1980).

RNA-DNA hybridization

The filters were prehybridized in 50% formamide, 5 × SSC ( l x SSC is 0.15M NaC1, 0.015M sodium acetate) at 42°C overnight, and then hybridized for 48 h to radiolabeled D N A probes labeled by the BRL nick-translation kit. The filters were washed with stepwise lowering of salt concentrations from i x SSC to 0.2 x SSC and raising the temperature from room temperature to 65°C. The filters were then exposed to X-ray film.

Probes

Rat fibronectin gene (obtained from D r .R .O .H yn es , MIT Center for Cancer Research, Cambridge, Mass.). The insert of rat fibronectin cDNA in Zrlfl was recloued into the EcoRI site of pGEM-2 pvLf-1 (Schwarzbauer et al. 1983).

Human fibronectin gene. The pHFN 2A12 plasmid was ob- tained from Dr. Ruoslahti, La Jolla Cancer Research Founda-

Table 1. Normalized mRNA contents in ataxia-telangiectasia (A-T) and hereditary hemorrhagic telangiectasia (HHT) fibroblasts relative to normal (N) fibroblasts. The density of the mRNA bands in each Northern blot was determined in a densitometer. In each case the density of the relevant mRNA band in the control was taken as 1.0, and the density of the same species of mRNA from the other fibro- blast species (A-T and HHT) were compared to 1.0

mRNA detected by probes

Human Rat Integrin ~3-Actin fibronectin fibronectin (4 kb) (1.6 kb) (8.6 kb) (8.6 kb)

N 1 1 1 1 A-T 0.2 0.3 1 2.2 HHT 1.1 1.1 2.5 6

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tion. The recombinant plasmid contains 380 bp of cDNA from the cell attachment site of the human fibronectin gene cloned in the PstI site of pBR322 (Oldberg et al. 1983).

Integrin gene. cDNA for chicken integrin band 3 (subunit) is called the pAX clone (Tamkun et al. 1986). It extends from the AccI site to the X m m I site. The insert has EcoRI- derived 12-mer linkers and was inserted into the EcoRI site of pGEM-2. The recombinant plasmid was received from Dr. R. O. Hynes.

The fi-actin gene. The recombinant plasmid pAC18.1 from rat (Nudel et al. 1983) was used.

Density determinations

The developed X-ray films were scanned in a densitometer. The density of each band was determined in arbitrary units for comparison between mRNA bands (Table 1).

Results

Expression of fibronectin genes in fibroblasts

Normal fibroblasts. The two fibroblast lines F-193 (Fig. 1A, lane 6) and GM 0037D were studied. Three fibronectin mRNA species were detected using the rat fibronectin probe: (1) a major band of 8.6kb, (2) mRNA ranging from 7.8 to 5.0 kb, and (3) a minor band of 3.5 kb (Fig. 1A, lane 6). Using the human fibronectin probe, only the 8.6 kb mRNA species was detected (Fig. 1B, lane 6). Similar results were obtained with RNA from GM 0037D fibroblasts (not shown).

Ataxia-telangiectasia fibroblasts. Two A-T fibroblast lines were studied with both rat and human probes. In line GM 5823 (Fig. 1A, lane 3) and F-28 (Fig. 1A, lane 4), the 8.6 kb fibronectin mRNA was detectable with the rat fibronectin probe, but at a reduced level compared to normal fibroblasts. The 3.5 kb mRNA was detected with the human fibronectin probe in RNA from HHT fibroblasts (Fig. 1B, lanes 1, 2). This probe did not detect the 3.5 kb mRNA in A-T fibroblasts (Fig. 1B, lanes 3, 4). The density of the 8.6kb mRNA band from A-T cells that was detected by the human fibronectin probe, was four- to fivefold lower than the same mRNA band in normal ceils (Table 1). The 5 and 3.5kb mRNA species were markedly reduced in A-T fibroblasts.

Fig.lA, B. Detection of fibronectin mRNA using rat fibronectin probe (A) and human fibronectin probe (B). The fibronectin mRNA was detected using the Northern blot technique. RNA in the different lanes was extracted from the following cell lines: 1 HHT (GM 3418), 2 HHT (GM 3419), 3 AT (GM 5823), 4 AT (F-28), 5 chick fibroblasts, 6 normal fibroblasts (F-193)

H H T fibroblasts. The two fibroblast cultures (GM 3419 and GM 3418; Fig. 1A, lanes 1, 2, respectively) resembled normal fibroblasts in having the three fibronectin mRNA species in the range of 8.6 to 5kb and 4 to 3.5kb. In densitometry studies (Table 1), the HHT fibronectin mRNA species re- sembled that of normal fibroblasts. The human fibronectin probe detected three mRNA species in HHT: the major mRNA species was the 8.6kb mRNA and the 5 and 3.5kb mRNAs appeared as distinct bands.

Chick embryo fibroblasts. These were included as controls for the detection of integrin mRNA. Figure 1A, lane 5 shows that the rat fibronectin gene probe detected only a faint mRNA band of 8.6 kb, while no fibronectin mRNA was detected with the human fibronectin probe in chick fibroblasts.

Fibronectin m R N A in A - T fibroblasts at low passage level

To assess the relationship between fibronectin mRNA syn- thesis in A-T fibroblasts and the physiological state of the cells, four A-T cultures (GM 5823, F-28, F-59 and F-320) were harvested at passage levels 9, 7, 9 and 8, respectively, and the

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fibronectin mRNA was hybridized with the human fibronectin DNA probe. In the normal fibroblast cultures F-193 and GM 0037D, the human fibronectin probe detected both the 8.6 and 5 kb mRNA species. In the A-T fibroblast cultures GM 5823 and F-320 an 8.6kb mRNA band was detected in addi- tion to the 5kb RNA. In A-T lines F-28 and F-59 the 8.6kb fibronectin RNA was not visible while the 5 kb RNA species was reduced but still detectable. Analysis of RNA from these four A-T fibroblast cultures at passage levels 11, 11, 13 and 12, respectively, revealed that all A-T fibroblasts had ceased synthesis of the 8.6kb fibronectin mRNA (not shown). The amount of 5 kb fibronectin in the mRNA was reduced and in F-28 fibroblasts fibronectin RNA was already not detectable at passage level 12. These results indicate that in the A-T fibroblasts the synthesis of fibronectin mRNA is shut-off at the time the cells approach senescence. The reason for the relatively early cessation of fibronectin mRNA synthesis in A- T fibroblasts is not yet known.

Expression of the integrin gene in normal and human fibroblasts

Chick embryo fibroblasts. The gene for one of the three poly- peptides that serve as the receptor (designated "integrin") for extracellular fibronectin and intracellular 13-actin fibers was originally isolated from chick embryo cells (Tamkun et al. 1986). These cells (Fig. 2, lane 5) were found to contain a large amount of mRNA with a mean size of about 4 kb. An additional mRNA band, of about 1.5kb, was also detected. The ratio between the 4kb mRNA and the 1.5 kb species re- vealed that the former was 15 times more abundant than the 1.5 kb mRNA. The size of integrin mRNA was previously re- ported to be 3.72 kb (Tamkun et al. 1986).

Normal human fibroblasts. Only the 4 kb mRNA band was de- tectable in normal human fibroblasts (Fig. 2, lane 6).

A-Tfibrobkasts. An mRNA band of about 4kb was also de- tectable in the two A-T fibroblast lines (Fig. 2, lanes 3, 4). When compared on the basis of densitometric values, to inte- grin mRNA in normal fibroblasts, the same amount of integ- rin mRNA was detected in A-T and normal fibroblasts (Table 1).

HHTfibroblasts. The 4 kb integrin mRNA was also detected in RNA extracted from the two HHT fibroblast lines (Fig. 2, lanes 1, 2). The amount of 4kb integrin mRNA in the HHT fibroblasts was 2.5 times higher than in normal fibroblasts (Table 1).

Expression of the fl-actin gene

All fibroblast lines contained the 1.6 kb mRNA species tran- scription product of the ~-actin gene. The amount of mRNA in A-T fibroblasts (Fig. 3, lanes 3, 4) was 2.2 times higher than that of normal fibroblasts (Fig. 3, lane 6). The contents of [3- actin mRNA detected in HHT fibroblast lines (Fig. 3, lanes 1, 2) was 2.5 times higher than in normal fibroblasts. The rat ~- actin DNA probe detected mRNA in chick embryo cells (Fig. 3, lane 5). The relative contents of [3-actin mRNA in the different fibroblast lines is presented in Table 1.

Fig. 2. Detection of integrin mRNA in cell cultures. The cloned inte- grin DNA probe was used in a Northern blot hybridization to detect integrin mRNA. The lane notations are as follows: I HHT (GM 3418), 2 HHT (GM 3419), 3 AT (GM 5823), 4 AT (F-28), 5 chick fibroblasts, 6 normal fibroblasts (F-193)

Fig. 3. Detection of [bactin mRNA in cell cultures. The cloned ~-actin DNA probe was used in a Northern blot hybridization to identify the 13-actin mRNA. The lane notations are as follows: 1 HHT (GM 3418), 2 HHT (GM 3419), 3 AT (GM 5823), 4 AT (F-28), 5 chick fibroblasts, 6 normal fibroblasts (F-193)

Discussion

Two observations led us to carry out the present study: (a) the evidence that the actin fibers in A-T fibroblasts are partly de- tached from the cell membrane (mentioned in Becker 1986; the original observation was made by Dr. R. Pollack, Colum- bia University, New York), while in normal fibroblasts the actin fibers are attached to the membrane; (b) the observation that the content of fibronectin (Mr 220 kDa, similar to plasma fibronectin) in the culture medium of A-T fibroblasts is higher than that of normal fibroblasts (Murnane and Painter 1983).

Three genes producing proteins that are involved in the maintenance of fibroblast structure have been described: (a) the gene for cellular fibronectin that attaches the fibroblast to

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the surface of the culture plate; (b) one of the two integrin genes coding for the two membrane glycoproteins to which the cellular fibronectin attaches extracellularly; (c) the gene for [3-actin which produces the actin filaments that attach intracellularly to the integrin glycoproteins. It is known that in A-T fibroblasts the actin fibers are partly detached from the cell membrane and that the morphology of the A-T fibroblast is affected (Becker 1986). In addition, since more fibronectin is released into the culture medium by A-T fibroblasts than by normal fibroblasts (Murnane and Painter 1983), it was hoped that by studying the expression of the three fibronectin genes which control cellular organization in A-T fibroblasts, the reason for the behavior of these cells in vitro might be eluci- dated.

The results summarized in Table 1 reveal that A-T fibro- blasts at a late passage in vitro make less fibronectin m R N A than either normal or HHT fibroblasts at a comparable pas- sage level. Expression of the integrin gene in A-T fibroblasts is similar to that in normal fibroblasts, while expression of the ~3-actin gene is higher. The lower content of fibronectin mRNA with a size less than 8.6kb and the marked reduc- tion or absence of the 3.5kb mRNA species suggests that the two fibronectin genes in the A-T fibroblasts may be in- tact and functional, producing unchanged amounts of mRNA during early passages in vitro; this expression is down-regu- lated when the fibroblasts near the end of their in vitro exis- tence, contrary to the regulation of the integrin and actin genes.

Three mRNA species (8.6, 8.0 and 7.7kb) for fibronectin in normal human fibroblasts were reported in fibroblast cell lines (HEL 299, Flow 7000, 8387) propagated in vitro (Col- ombi et al. 1986). Other cell lines had 8.0, 8.2, 8.4, 7.8 or 7.7kb mRNA species, and most often two m R N A species per cell. In our study, the mRNA species in normal fibroblasts were 8.6, - 5 and 3.5 kb in size. Similar m R N A species were found to exist in HHT fibroblasts. In A-T, only the 8.6kb mRNA species of fibronectin was detected, but at a lower level compared to the controls.

The presence of several mRNA species for fibronectin indicates that m R N A splicing is a dominant feature in the transcription of fibronectin genes; both fibronectin genes are involved in the splicing (Oldberg and Ruoslahti 1986). Yet the gene for cellular fibronectin is more sensitive to viral trans- formation and is shut off in Rous sarcoma virus-transformed fibroblasts (Colombi et al. 1986).

Since A-T fibroblasts were found to produce fibronectin and release it from the cells into the culture medium (Mur- nane and Painter 1983; D.M.Tatsuka , personal communica- tion), the released fibronectin is most probably plasma fibro- nectin, the product of the fibronectin gene in chromosome 2. Thus the --5 kb and 3.5 kb mRNA species detectable in A-T fibroblasts by the human fibronectin gene probe should be the transcript of the plasma fibronectin gene located in chromo- some 2. However, the early decline of the 8.6 kb fibronectin mRNA in A-T fibroblasts at higher passage levels might sug- gest that it originates from the cellular fibronectin gene in chromosome 11 (q12.1-q13.5, designated FNL2, i.e. fibro- nectin-like 2; McKusick 1986). The findings by Murnane and Painter (1983) and D.M.Ta tsuka (personal communication) that the fibronectin content in the medium of A-T ceils is higher than that of normal cells might be explained by the sta- bility of the protein product compared to the short half-life of the mRNA.

It is possible that the down-regulation of the cellular fibro- nectin gene in A-T fibroblasts (detectable by the probe of human fibronectin D N A containing the cell attachment se- quence) indicates the decline of a regulatory mechanism in A-T cells as they approach senescence. The finding that mRNA transcription from the integrin and 13-actin genes in A-T fibro- blasts remains unchanged at late passage level suggests that these genes are not affected by the mechanism that leads to the shut-off of, presumably, the cellular fibronectin gene. Thus the function of the fibronectin gene may be connected directly or indirectly with that of the gene in A-T fibroblasts. It is possible that the reduced level of fibronectin mRNA in A-T fibroblasts is associated with the early senescence of the cells which have a short life-span in vitro. Alternatively, the short life-span of these cells might result from the reduced ex- pression of cellular fibronectin. Studies on the expression of the fibronectin gene in fibroblasts from the genetic disorder, progeria, are planned.

If the function of the cellular fibronectin gene is affected in the A-T condition, and is controlled by the A-T gene, could this defect of the fibronectin gene cause the numerous pleio- tropic effects reported to occur in A-T homozygotes? Indeed, the abnormal development (hypoplasia) of organs such as the thymus and ovaries, and growth retardation in A-T patients might be explained on the basis of defective synthesis of cellu- lar fibronectin by the connective tissue fibroblasts. This func- tion is necessary for the embryonic genesis of certain organs (Oldberg et al. 1983).

A-T homozygous patients are prone to lymphoreticular neoplasia, while obligate heterozygotes have a high incidence of cancer in many organs (Swift et al. 1976). It is of interest that two of the genes close to the cellular fibronectin gene on chromosome 11 are connected with neoplasia: the oncogene INT2 (in 11q13) and B-cell leukemia 1 (bcl-1) oncogene (in 11q13.3) (McKusick 1986). It is unclear what role, if any, these oncogenes might have in the development of lympho- reticular neoplasia in A-T homozygotes or heterozygotes, or whether a mutation in the cellular fibronectin gene may affect the activity of these proto-oncogenes.

The hypersensitivity of A-T fibroblasts to X-rays (Painter 1985), radiomimetic drugs like neocarzinostatin (NCS) and bleomycin (Shiloh and Becker 1982; Shaham et al. 1983; Shiloh et al. 1983a, b, 1985a), as well as a tumor promoter (Shiloh et al. 1985b) may also be explained as resulting from the reduced synthesis of cellular fibronectin. The absence of the latter could lead to the inability of the A-T fibroblasts to form colonies and to a limited life-span in vitro (Shiloh et al. 1982). Treatment of A-T fibroblasts with epidermal growth factor (EGF) was found to increase the plating efficiency of the fibroblasts (Shiloh et al. 1982). It is of interest that E G F has been reported to activate the synthesis of cellular fibro- nectin in human fibroblasts (Chen et al. 1987).

Comparison of the expression of fibronectin, integrin and J3-actin in A-T fibroblasts with that in fibroblasts from two patients with HHT, showed that although telangiectasia and brain damage are features common to both genetic disorders, HHT fibroblasts differed markedly from A-T fibroblasts in having a high level of fibronectin and integrin mRNA expres- sion at late passage levels in vitro. [3-actin mRNA contents were similar in fibroblasts from patients with A-T and HHT and were higher than in normal fibroblasts. Thus, the gene re- sponsible for HHT is different from that of A-T. It is possible that regulation of the gene in chromosome 3 is affected in

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H H T , while regula t ion of the gene for cellular f ibronect in in c h r o m o s o m e 11 might be affected in the A - T syndrome.

Acknowledgements. This study was supported by grants from the United States-Israel Binational Science Foundation (BSF), Jerusalem, Israel in collaboration with Dr. L. Schultz, The Jackson Laboratories, Bar Harbor, Me., and from the Fritz Thyssen Foundation, FRG. The authors are indebted to Dr. R. Hynes, Center for Cancer Research, MIT, Cambridge, Mass., for the plasmids containing rat fibronectin and chicken integrin DNAs, to Dr.E.Ruoslahti, Cancer Research Center, La Jolla Cancer Research Foundation, La Jolla, Calif., for the recombinant plasmid containing the cell attachment domain of human fibronectin DNA, and to Dr.H. Cedar for the recombinant plasmid with the ]3-actin gene. The support of the Foundation for the Study of Molecular Virology and Cell Biology, Phoenix, Ariz., is gratefully acknowledged. The authors thank Dr.M.Tatsuka, The Research Institute of Microbial Diseases, Osaka University, Japan, for providing the results of his studies on A-T.

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Received May 1, 1988 / Revised July 18, 1988