POSSIBLE INTERACTION OF CELL MEMBRANE- BOUND N-ras PATHWAYS WITH NF2-RELATED CYTOSKELETON FACTORS IN ONCOGENESIS OF MENINGIOMAS
Yapijakis C1, Mamali I1, Papapetrou KP2, Stranjalis GS2, Protopapa DP3, Vassilopoulos D1, Sakas DE2
*Corresponding Author: Christos Yapijakis, D.M.D., M.S., Ph.D., Department of Neurology, University of Athens Medical School, Eginition Hospital, Vas Sofias 74, Athens 11528, Greece; Tel: +30-10-7289-125; Fax: +30-10-8811-243; E-mail: cyapijakis_ua_gr@yahoo.com
page: 17

MATERIALS AND METHODS

The studied material consisted of 14 meningiomas (10 frozen and four paraffin-embedded tissue samples) obtained from 14 unrelated Greek patients who had been operated on at the Evangelismos Hospital, Athens, Greece. The patients were mostly women (n = 8), with an age range of 37-72 years (median 52 years), and a family history free of other cases of meningioma or NF2. Three of the meningiomas were of grade I, eight of grade II and three of grade III (Table 1). The tumor samples were assigned a number and examined blindly.

Ten biopsies were frozen in isopentane, cooled in liquid nitrogen, and cryostat 6 mm sections were immunostained by a standard avidin-biotin-peroxidase technique (see below). In parallel, a sural nerve biopsy (obtained from a neuropathy patient without any histopathological findings) was analyzed as a control for immunostaining. The staining of each sample was densitometrically compared to that of the control, and considered to be (i.e., at least 10X less intensity), + (i.e., about equal intensity), ++ or +++ (i.e., at least 10X r 100X more intensity, respectively). Briefly, the immunostaining method included:

 

1.   Incubation with normal rabbit serum diluted 1/5 with phosphate buffer solution (PBS) for 10 minutes.

2.   Incubation with primary antibody c-N-ras IgG1 F155-227 (OncogeneO; Research Products, Boston, MA, USA) in 1/50 dilution for 1 hour and washing with PBS.

3.   Incubation with a biotinylated F(ab)2 fragment of anti-mouse IgG in 1/20 dilution for 30 minutes and washing with PBS.

4.   Incubation with extravidin-conjugated peroxidase in 1/20 dilution for 30 minutes and washing with PBS.

5.   Incubation with chromogen 3-amino-9-ethylcarbazole.

6.   Examination and intensity analysis under a light microscope, and photographic documentation.

Sections from four fixed, wax-embedded tissue samples were cut at 6 mm and dried at room temperature. The sections were then de-waxed with three washes of xylene, washed three times with alcohol, and finally submerged in water. Then, the immunostaining method was applied.

Total DNA was isolated from a region of each menin­gioma sample immediately adjunct to the sections taken. The DNA extraction was performed using a Nucleon TissueO kit (Amersham Pharmacia Biotech, Little Chalfont, Buckinghamshire, UK). A 200 bp fragment of exon 2 and a 76 bp fragment of exon 4 (including hot spot codons 57 and 198, respectively) were amplified under standard polymerase chain reaction (PCR) conditions. The sequences of the primers used [12] were as follows: for the codon 57 fragment (annealing temperature 60C) S1 5-TTGCTCACAGTG TCCTTCCC-3 and S2 5-TCAGCCCCACCAGTTTC ATC-3; and for the codon 198 fragment (annealing temperature 62C) C1 5-GACTCCGGAAATGTGGGAG GA-3 and C2 5-GATTGGGCCTCACCT GGCTC-3.

The PCR products were digested with the restriction enzyme DdeI and analyzed by agarose gel electrophoresis. Digested products of 109+91 bp, and 56+20 bp were detected only when the CGA codons was mutated to TGA (stop codon).

 

 

 

 

 

 

 

Figure 1. Molecular detection of mutations at hot spot codon 57 of the NF2 gene (see text for details). Lane 1: molecular weight marker; lane 2: undigested PCR product of a normal control; lane 3: negative PCR control (without DNA); lanes 4-8: DdeI-treated PCR products of five meningioma samples. Samples 4 and 5 have an undigested DNA fragment of 200 bp (normal homozygotes at codon 57), while samples 6-8 (5065, 5436 and 644) are heterozy­gotes for the normal 200 bp allele and the mutant allele of 109+91 bp.

 

 

 

 

Sample Number

 

Grade

 

Sex-Age

 

NF2

 

N-ras

 

264

 

I

 

F-43

 

wt

 

[+]

 

468 (p)

 

II

 

M-43

 

m

 

[++]

 

513

 

III

 

M-68

 

wt

 

[+++]

 

644 (p)

 

III

 

M-70

 

m

 

[++]

 

1530

 

II

 

F-72

 

wt

 

[+++]

 

3050

 

II

 

F-52

 

wt

 

[++]

 

3806

 

II

 

F-45

 

wt

 

[+]

 

4659 (p)

 

II

 

F-37

 

wt

 

[+]

 

4688

 

I

 

F-52

 

wt

 

[+]

 

4959

 

II

 

F-58

 

wt

 

[++]

 

5065

 

I

 

M-65

 

m

 

[]

 

5436 (p)

 

II

 

F-50

 

m

 

[++]

 

10333

 

III

 

F-69

 

wt

 

[++]

 

11507

 

II

 

F-49

 

wt

 

[++]

 

F = female; M = male; (p) paraffin-embedded tissue; (wt) homozygous wild-type allele; (m) mutant allele in the heterozygous state; [+] staining intensity comparable to control; [] staining intensity less than control; [++]/[+++] staining intensity more than control.

 

Table 1. Results of immunohistochemical detection of N-ras protein and molecular detection of mutant NF2 gene in meningioma samples

 

 




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