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Methylation and silencing of the retinoic acid receptor-beta 2 gene in cervical cancer.

Ivanova T, Petrenko A, Gritsko T, Vinokourova S, Eshilev E, Kobzeva V, Kisseljov F, Kisseljova N - BMC Cancer (2002)

Bottom Line: In 8 out 20 cervical SCC (40%) the levels of RAR-beta2 mRNA were decreased or undetectable in comparison with non-neoplastic cervix tissues.These findings suggest that methylation of the 5' region of RAR-beta2 gene may contribute to gene silencing and that methylation of this region may be an important and early event in cervical carcinogenesis.These findings may be useful to make retinoids more effective as preventive and therapeutic agents in combination with inhibitors of DNA methylation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Carcinogenesis, Cancer Research Center, Kashirskoye shosse 24, Moscow 115478, Russia. tan_ivanova@mail.ru

ABSTRACT

Background: Expression of the retinoic acid receptor beta2 (RAR-beta2), a putative tumor suppressor gene, is reduced in various human cancers, including squamous cell carcinomas (SCC) of the uterine cervix. The mechanism of the inhibition of RAR-beta2 expression remains obscure. We examined whether methylation of RAR-beta2 gene could be responsible for this silencing in cervical SCC.

Methods: Expression of RAR-beta2 mRNA and methylation status of the 5' region of RAR-beta2 gene were examined in 20 matched specimens from patients with cervical SCC and in three cervical cancer cell lines by Northern blot analysis and methylation-specific PCR (MSP) assay or Southern blot analysis respectively.

Results: In 8 out 20 cervical SCC (40%) the levels of RAR-beta2 mRNA were decreased or undetectable in comparison with non-neoplastic cervix tissues. All 8 tumors with reduced levels of RAR-beta2 mRNA expression showed methylation of the promoter and the first exon expressed in the RAR-beta2 transcript. The RAR-beta2 gene from non-neoplastic cervical tissues was mostly unmethylated and expressed, but methylated alleles of the gene were found in three samples of the morphologically normal tissues adjacent to the tumors. Three cervical cancer cell lines with extremely low level of RAR-beta2 mRNA expression, SiHA, HeLA and CaSki, also showed methylation of this region of the RAR-beta2 gene.

Conclusions: These findings suggest that methylation of the 5' region of RAR-beta2 gene may contribute to gene silencing and that methylation of this region may be an important and early event in cervical carcinogenesis. These findings may be useful to make retinoids more effective as preventive and therapeutic agents in combination with inhibitors of DNA methylation.

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A. Restriction map of promoter-exon region of RARβ2 gene for methylation-sensitive Hpa II enzyme. The bolt line shows the 680 bp DNA probe generated by PCR of the promoter and exon 3. The expected fragment sizes which are recognized by this probe are shown in the bottom part of the map. The transcription start site is indcated by broken arrow. The positions of the primers for MSP assay are indicated by arrows. The 146 bp fragment is the region analyzed for CpG methylation by MSP assay. The triangles show the positions of two RAREs within the promoter region. H – HpaII sites, the positions of the Hpa II sites in relation to the trascription start site are indicated by fugures in bp. B. Northern blot analysis of total RNA isolated from cervical carcinomas (T) and adjacent cervical normal tissues (N). The blots were hybridized with 32P labeled DNA probes for RARβ2 and GAPDH. Lanes1, 2 – 4N, 4T, lanes 3, 4 – 5N, 5T, lanes 5,6 – 7N, 7T respectively. Case numbers correspond to that indicated in table 1. The arrows show the positions of 28S and 18S RNA. C. Southern blot analysis of DNA isolated from cervical carcinomas (T) and adjacent cervical normal tissues (N). The blots were hybridized with 680 bp 32P labeled DNA probe for RARβ2. The bold arrow shows the positions of a minimal 1.1 kbp fragment, recognized by this probe. Lanes 1, 3, 5, 7, 9, 11, 13, 15 – DNA digested by MspI; lanes 2, 4, 6, 8, 10, 12, 14, 16 – DNA digested by HpaII; lanes 1, 2 – 9N; lanes 3, 4 – 9T; lanes 5,6 – 4N; lanes 7, 8 – 4T; lanes 9, 10 – 7N; lanes 11,12 – 7T; lanes 13,14 – SiHa; lanes 15, 16 – HeLa. Case numbers correspond to that indicated in table 1. D. MSP assay of DNA isolated from cervical carcinomas (T), adjacent cervical normal tissues (N) and leucocytes (L). All DNA samples were treated with sodium bisulfite before amplification of a 146 bp fragment with methylation-specific primers (M) and unmethylation-specific primers (U). Sample numbers on the top of the gel correspond to that indicated in table 1; mr – molecular weight marker.
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Figure 1: A. Restriction map of promoter-exon region of RARβ2 gene for methylation-sensitive Hpa II enzyme. The bolt line shows the 680 bp DNA probe generated by PCR of the promoter and exon 3. The expected fragment sizes which are recognized by this probe are shown in the bottom part of the map. The transcription start site is indcated by broken arrow. The positions of the primers for MSP assay are indicated by arrows. The 146 bp fragment is the region analyzed for CpG methylation by MSP assay. The triangles show the positions of two RAREs within the promoter region. H – HpaII sites, the positions of the Hpa II sites in relation to the trascription start site are indicated by fugures in bp. B. Northern blot analysis of total RNA isolated from cervical carcinomas (T) and adjacent cervical normal tissues (N). The blots were hybridized with 32P labeled DNA probes for RARβ2 and GAPDH. Lanes1, 2 – 4N, 4T, lanes 3, 4 – 5N, 5T, lanes 5,6 – 7N, 7T respectively. Case numbers correspond to that indicated in table 1. The arrows show the positions of 28S and 18S RNA. C. Southern blot analysis of DNA isolated from cervical carcinomas (T) and adjacent cervical normal tissues (N). The blots were hybridized with 680 bp 32P labeled DNA probe for RARβ2. The bold arrow shows the positions of a minimal 1.1 kbp fragment, recognized by this probe. Lanes 1, 3, 5, 7, 9, 11, 13, 15 – DNA digested by MspI; lanes 2, 4, 6, 8, 10, 12, 14, 16 – DNA digested by HpaII; lanes 1, 2 – 9N; lanes 3, 4 – 9T; lanes 5,6 – 4N; lanes 7, 8 – 4T; lanes 9, 10 – 7N; lanes 11,12 – 7T; lanes 13,14 – SiHa; lanes 15, 16 – HeLa. Case numbers correspond to that indicated in table 1. D. MSP assay of DNA isolated from cervical carcinomas (T), adjacent cervical normal tissues (N) and leucocytes (L). All DNA samples were treated with sodium bisulfite before amplification of a 146 bp fragment with methylation-specific primers (M) and unmethylation-specific primers (U). Sample numbers on the top of the gel correspond to that indicated in table 1; mr – molecular weight marker.

Mentions: The level of the expression of RAR-β2 mRNA was evaluated in 20 samples of invasive squamous cell carcinomas of uterine cervix at low and moderate levels of differentiation and in the morphologically normal tumor-adjacent tissues of cervix in order to select a group of tumors with deficient expression of RAR-β2 (Figure 1B, Table 1). The level of RAR-β2 mRNA expression was significantly decreased in 8 out of 20 tumors constituting 5 – 48% of the expression level of the corresponding morphologically normal tissue. An extremely low (undetectable) level of mRNA expression was observed in one tumor, as well as in the adjacent morphologically normal tissue (Fig. 1B, lanes 5,6; Table 1, case 7). Thus, in 40% of cervical SCC the expression of the RAR-β2 mRNA was significantly decreased. An extremely low level of mRNA expression, wich was observed earlier in three cell lines HeLA, SiHa and CaSki [22], was confirmed in our experiment (data are not presented).


Methylation and silencing of the retinoic acid receptor-beta 2 gene in cervical cancer.

Ivanova T, Petrenko A, Gritsko T, Vinokourova S, Eshilev E, Kobzeva V, Kisseljov F, Kisseljova N - BMC Cancer (2002)

A. Restriction map of promoter-exon region of RARβ2 gene for methylation-sensitive Hpa II enzyme. The bolt line shows the 680 bp DNA probe generated by PCR of the promoter and exon 3. The expected fragment sizes which are recognized by this probe are shown in the bottom part of the map. The transcription start site is indcated by broken arrow. The positions of the primers for MSP assay are indicated by arrows. The 146 bp fragment is the region analyzed for CpG methylation by MSP assay. The triangles show the positions of two RAREs within the promoter region. H – HpaII sites, the positions of the Hpa II sites in relation to the trascription start site are indicated by fugures in bp. B. Northern blot analysis of total RNA isolated from cervical carcinomas (T) and adjacent cervical normal tissues (N). The blots were hybridized with 32P labeled DNA probes for RARβ2 and GAPDH. Lanes1, 2 – 4N, 4T, lanes 3, 4 – 5N, 5T, lanes 5,6 – 7N, 7T respectively. Case numbers correspond to that indicated in table 1. The arrows show the positions of 28S and 18S RNA. C. Southern blot analysis of DNA isolated from cervical carcinomas (T) and adjacent cervical normal tissues (N). The blots were hybridized with 680 bp 32P labeled DNA probe for RARβ2. The bold arrow shows the positions of a minimal 1.1 kbp fragment, recognized by this probe. Lanes 1, 3, 5, 7, 9, 11, 13, 15 – DNA digested by MspI; lanes 2, 4, 6, 8, 10, 12, 14, 16 – DNA digested by HpaII; lanes 1, 2 – 9N; lanes 3, 4 – 9T; lanes 5,6 – 4N; lanes 7, 8 – 4T; lanes 9, 10 – 7N; lanes 11,12 – 7T; lanes 13,14 – SiHa; lanes 15, 16 – HeLa. Case numbers correspond to that indicated in table 1. D. MSP assay of DNA isolated from cervical carcinomas (T), adjacent cervical normal tissues (N) and leucocytes (L). All DNA samples were treated with sodium bisulfite before amplification of a 146 bp fragment with methylation-specific primers (M) and unmethylation-specific primers (U). Sample numbers on the top of the gel correspond to that indicated in table 1; mr – molecular weight marker.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC101392&req=5

Figure 1: A. Restriction map of promoter-exon region of RARβ2 gene for methylation-sensitive Hpa II enzyme. The bolt line shows the 680 bp DNA probe generated by PCR of the promoter and exon 3. The expected fragment sizes which are recognized by this probe are shown in the bottom part of the map. The transcription start site is indcated by broken arrow. The positions of the primers for MSP assay are indicated by arrows. The 146 bp fragment is the region analyzed for CpG methylation by MSP assay. The triangles show the positions of two RAREs within the promoter region. H – HpaII sites, the positions of the Hpa II sites in relation to the trascription start site are indicated by fugures in bp. B. Northern blot analysis of total RNA isolated from cervical carcinomas (T) and adjacent cervical normal tissues (N). The blots were hybridized with 32P labeled DNA probes for RARβ2 and GAPDH. Lanes1, 2 – 4N, 4T, lanes 3, 4 – 5N, 5T, lanes 5,6 – 7N, 7T respectively. Case numbers correspond to that indicated in table 1. The arrows show the positions of 28S and 18S RNA. C. Southern blot analysis of DNA isolated from cervical carcinomas (T) and adjacent cervical normal tissues (N). The blots were hybridized with 680 bp 32P labeled DNA probe for RARβ2. The bold arrow shows the positions of a minimal 1.1 kbp fragment, recognized by this probe. Lanes 1, 3, 5, 7, 9, 11, 13, 15 – DNA digested by MspI; lanes 2, 4, 6, 8, 10, 12, 14, 16 – DNA digested by HpaII; lanes 1, 2 – 9N; lanes 3, 4 – 9T; lanes 5,6 – 4N; lanes 7, 8 – 4T; lanes 9, 10 – 7N; lanes 11,12 – 7T; lanes 13,14 – SiHa; lanes 15, 16 – HeLa. Case numbers correspond to that indicated in table 1. D. MSP assay of DNA isolated from cervical carcinomas (T), adjacent cervical normal tissues (N) and leucocytes (L). All DNA samples were treated with sodium bisulfite before amplification of a 146 bp fragment with methylation-specific primers (M) and unmethylation-specific primers (U). Sample numbers on the top of the gel correspond to that indicated in table 1; mr – molecular weight marker.
Mentions: The level of the expression of RAR-β2 mRNA was evaluated in 20 samples of invasive squamous cell carcinomas of uterine cervix at low and moderate levels of differentiation and in the morphologically normal tumor-adjacent tissues of cervix in order to select a group of tumors with deficient expression of RAR-β2 (Figure 1B, Table 1). The level of RAR-β2 mRNA expression was significantly decreased in 8 out of 20 tumors constituting 5 – 48% of the expression level of the corresponding morphologically normal tissue. An extremely low (undetectable) level of mRNA expression was observed in one tumor, as well as in the adjacent morphologically normal tissue (Fig. 1B, lanes 5,6; Table 1, case 7). Thus, in 40% of cervical SCC the expression of the RAR-β2 mRNA was significantly decreased. An extremely low level of mRNA expression, wich was observed earlier in three cell lines HeLA, SiHa and CaSki [22], was confirmed in our experiment (data are not presented).

Bottom Line: In 8 out 20 cervical SCC (40%) the levels of RAR-beta2 mRNA were decreased or undetectable in comparison with non-neoplastic cervix tissues.These findings suggest that methylation of the 5' region of RAR-beta2 gene may contribute to gene silencing and that methylation of this region may be an important and early event in cervical carcinogenesis.These findings may be useful to make retinoids more effective as preventive and therapeutic agents in combination with inhibitors of DNA methylation.

View Article: PubMed Central - HTML - PubMed

Affiliation: Institute of Carcinogenesis, Cancer Research Center, Kashirskoye shosse 24, Moscow 115478, Russia. tan_ivanova@mail.ru

ABSTRACT

Background: Expression of the retinoic acid receptor beta2 (RAR-beta2), a putative tumor suppressor gene, is reduced in various human cancers, including squamous cell carcinomas (SCC) of the uterine cervix. The mechanism of the inhibition of RAR-beta2 expression remains obscure. We examined whether methylation of RAR-beta2 gene could be responsible for this silencing in cervical SCC.

Methods: Expression of RAR-beta2 mRNA and methylation status of the 5' region of RAR-beta2 gene were examined in 20 matched specimens from patients with cervical SCC and in three cervical cancer cell lines by Northern blot analysis and methylation-specific PCR (MSP) assay or Southern blot analysis respectively.

Results: In 8 out 20 cervical SCC (40%) the levels of RAR-beta2 mRNA were decreased or undetectable in comparison with non-neoplastic cervix tissues. All 8 tumors with reduced levels of RAR-beta2 mRNA expression showed methylation of the promoter and the first exon expressed in the RAR-beta2 transcript. The RAR-beta2 gene from non-neoplastic cervical tissues was mostly unmethylated and expressed, but methylated alleles of the gene were found in three samples of the morphologically normal tissues adjacent to the tumors. Three cervical cancer cell lines with extremely low level of RAR-beta2 mRNA expression, SiHA, HeLA and CaSki, also showed methylation of this region of the RAR-beta2 gene.

Conclusions: These findings suggest that methylation of the 5' region of RAR-beta2 gene may contribute to gene silencing and that methylation of this region may be an important and early event in cervical carcinogenesis. These findings may be useful to make retinoids more effective as preventive and therapeutic agents in combination with inhibitors of DNA methylation.

Show MeSH
Related in: MedlinePlus