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Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets.

Sousa JF, Espreafico EM - BMC Cancer (2008)

Bottom Line: We confirmed the differential expression of all genes selected for validation.In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking.A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular and Molecular Biology and Pathogenic Bioagents of Faculty of Medicine of Ribeirão Preto - University of São Paulo, Ribeirão Preto, SP, Brazil. jdfsousa@gmail.com

ABSTRACT

Background: Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of alphavbeta3-integrin and low levels of RHOC.

Methods: Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified.

Results: We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library.

Conclusion: This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.

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KISS1 and RHOC mRNA expression in a panel of RGP, VGP and metastatic melanoma cell lines. Comparison of the expression levels of the KISS1 metastasis suppressor gene (A) and the small GTPase RHOC (B) among melanoma cell lines of different stages of tumor progression supported the selection of WM1552C cell line as the RGP representative for suppression subtractive hybridization against a pool of metastatic cell lines. Samples of 20 μg of total RNA from different melanoma cell lines were submitted to electrophoresis in 1% agarose-formaldehyde gel and transferred to nylon membrane (Hybond N, Amersham Pharmacia Biotech) by standard methods. Fragments of the indicated genes were radiolabeled with [α-32P]-dCTP by random-priming (Rad-prime kit, Invitrogen) and used as probes for Northern blot hybridization. In order to correct for loading differences, after stripping, the blots were probed with a ACTB (β-actin) cDNA fragment.
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Figure 1: KISS1 and RHOC mRNA expression in a panel of RGP, VGP and metastatic melanoma cell lines. Comparison of the expression levels of the KISS1 metastasis suppressor gene (A) and the small GTPase RHOC (B) among melanoma cell lines of different stages of tumor progression supported the selection of WM1552C cell line as the RGP representative for suppression subtractive hybridization against a pool of metastatic cell lines. Samples of 20 μg of total RNA from different melanoma cell lines were submitted to electrophoresis in 1% agarose-formaldehyde gel and transferred to nylon membrane (Hybond N, Amersham Pharmacia Biotech) by standard methods. Fragments of the indicated genes were radiolabeled with [α-32P]-dCTP by random-priming (Rad-prime kit, Invitrogen) and used as probes for Northern blot hybridization. In order to correct for loading differences, after stripping, the blots were probed with a ACTB (β-actin) cDNA fragment.

Mentions: For selecting the cell lines, we checked on the expression of three known molecular markers of melanoma progression, KISS1 and RHOC mRNAs and the αvβ3 integrin, in the panel of melanoma cell lines used here. KISS1 mRNA expression was detected by Northern blot only in the RGP cell line WM1552C (Fig. 1A). So even the other two RGP cell lines (WM35 and WM1789) included in our study failed to show detectable levels of KISS1 mRNA, although by using a more sensitive method (RT-PCR/Southern blotting), a weak expression of KISS1 transcript in the WM35 cell line in contrast with lack of expression in WM793 (VGP) and 1205Lu (metastatic) was previously reported [21].


Suppression subtractive hybridization profiles of radial growth phase and metastatic melanoma cell lines reveal novel potential targets.

Sousa JF, Espreafico EM - BMC Cancer (2008)

KISS1 and RHOC mRNA expression in a panel of RGP, VGP and metastatic melanoma cell lines. Comparison of the expression levels of the KISS1 metastasis suppressor gene (A) and the small GTPase RHOC (B) among melanoma cell lines of different stages of tumor progression supported the selection of WM1552C cell line as the RGP representative for suppression subtractive hybridization against a pool of metastatic cell lines. Samples of 20 μg of total RNA from different melanoma cell lines were submitted to electrophoresis in 1% agarose-formaldehyde gel and transferred to nylon membrane (Hybond N, Amersham Pharmacia Biotech) by standard methods. Fragments of the indicated genes were radiolabeled with [α-32P]-dCTP by random-priming (Rad-prime kit, Invitrogen) and used as probes for Northern blot hybridization. In order to correct for loading differences, after stripping, the blots were probed with a ACTB (β-actin) cDNA fragment.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2267200&req=5

Figure 1: KISS1 and RHOC mRNA expression in a panel of RGP, VGP and metastatic melanoma cell lines. Comparison of the expression levels of the KISS1 metastasis suppressor gene (A) and the small GTPase RHOC (B) among melanoma cell lines of different stages of tumor progression supported the selection of WM1552C cell line as the RGP representative for suppression subtractive hybridization against a pool of metastatic cell lines. Samples of 20 μg of total RNA from different melanoma cell lines were submitted to electrophoresis in 1% agarose-formaldehyde gel and transferred to nylon membrane (Hybond N, Amersham Pharmacia Biotech) by standard methods. Fragments of the indicated genes were radiolabeled with [α-32P]-dCTP by random-priming (Rad-prime kit, Invitrogen) and used as probes for Northern blot hybridization. In order to correct for loading differences, after stripping, the blots were probed with a ACTB (β-actin) cDNA fragment.
Mentions: For selecting the cell lines, we checked on the expression of three known molecular markers of melanoma progression, KISS1 and RHOC mRNAs and the αvβ3 integrin, in the panel of melanoma cell lines used here. KISS1 mRNA expression was detected by Northern blot only in the RGP cell line WM1552C (Fig. 1A). So even the other two RGP cell lines (WM35 and WM1789) included in our study failed to show detectable levels of KISS1 mRNA, although by using a more sensitive method (RT-PCR/Southern blotting), a weak expression of KISS1 transcript in the WM35 cell line in contrast with lack of expression in WM793 (VGP) and 1205Lu (metastatic) was previously reported [21].

Bottom Line: We confirmed the differential expression of all genes selected for validation.In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking.A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Cellular and Molecular Biology and Pathogenic Bioagents of Faculty of Medicine of Ribeirão Preto - University of São Paulo, Ribeirão Preto, SP, Brazil. jdfsousa@gmail.com

ABSTRACT

Background: Melanoma progression occurs through three major stages: radial growth phase (RGP), confined to the epidermis; vertical growth phase (VGP), when the tumor has invaded into the dermis; and metastasis. In this work, we used suppression subtractive hybridization (SSH) to investigate the molecular signature of melanoma progression, by comparing a group of metastatic cell lines with an RGP-like cell line showing characteristics of early neoplastic lesions including expression of the metastasis suppressor KISS1, lack of alphavbeta3-integrin and low levels of RHOC.

Methods: Two subtracted cDNA collections were obtained, one (RGP library) by subtracting the RGP cell line (WM1552C) cDNA from a cDNA pool from four metastatic cell lines (WM9, WM852, 1205Lu and WM1617), and the other (Met library) by the reverse subtraction. Clones were sequenced and annotated, and expression validation was done by Northern blot and RT-PCR. Gene Ontology annotation and searches in large-scale melanoma expression studies were done for the genes identified.

Results: We identified 367 clones from the RGP library and 386 from the Met library, of which 351 and 368, respectively, match human mRNA sequences, representing 288 and 217 annotated genes. We confirmed the differential expression of all genes selected for validation. In the Met library, we found an enrichment of genes in the growth factors/receptor, adhesion and motility categories whereas in the RGP library, enriched categories were nucleotide biosynthesis, DNA packing/repair, and macromolecular/vesicular trafficking. Interestingly, 19% of the genes from the RGP library map to chromosome 1 against 4% of the ones from Met library.

Conclusion: This study identifies two populations of genes differentially expressed between melanoma cell lines from two tumor stages and suggests that these sets of genes represent profiles of less aggressive versus metastatic melanomas. A search for expression profiles of melanoma in available expression study databases allowed us to point to a great potential of involvement in tumor progression for several of the genes identified here. A few sequences obtained here may also contribute to extend annotated mRNAs or to the identification of novel transcripts.

Show MeSH
Related in: MedlinePlus