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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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Validation by Northern blot and RT-PCR of the expression pattern of seven genes identified in the SSH libraries. Frames depict the names of cell lines used in the construction of the libraries. The inserts of cDNA clones corresponding to the genes DCN (decorin) (A), ALS2CR7 (B) and MBOAT1 (C) of the RGP library; YWHAZ (14-3-3 ξ) (D) identified in both libraries; and MITF (E) and PLP1 (F) from the Met library were isolated and used as probes for hybridization in Northern blots containing total RNA from the melanoma cell lines indicated above the panels – Blank lanes mean that the corresponding cell line was not included in the Northern blot, and were introduced to allow alignment among panels. Northern blots were prepared as described in Figure 1. HLA-DRA (G) identified in the Met library was validated by RT-PCR. For RT-PCR, total RNA samples (2 μg) from the indicated cell lines were, after DNase treatment, submitted to reverse transcription with Superscript II (Invitrogen) using oligo dT as primer and the cDNA was used as template for PCR amplification with HLA-DRA primers. After 25, 28, 30 and 32 amplification cycles, 5 μl aliquots were collected for agarose gel electrophoresis. As endogenous control, a pair of primers for the ACTB (β-actin) mRNA was used. C: Control RT-PCR amplification using as template RNA (DNase treated) without prior reverse transcription.
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Figure 3: Validation by Northern blot and RT-PCR of the expression pattern of seven genes identified in the SSH libraries. Frames depict the names of cell lines used in the construction of the libraries. The inserts of cDNA clones corresponding to the genes DCN (decorin) (A), ALS2CR7 (B) and MBOAT1 (C) of the RGP library; YWHAZ (14-3-3 ξ) (D) identified in both libraries; and MITF (E) and PLP1 (F) from the Met library were isolated and used as probes for hybridization in Northern blots containing total RNA from the melanoma cell lines indicated above the panels – Blank lanes mean that the corresponding cell line was not included in the Northern blot, and were introduced to allow alignment among panels. Northern blots were prepared as described in Figure 1. HLA-DRA (G) identified in the Met library was validated by RT-PCR. For RT-PCR, total RNA samples (2 μg) from the indicated cell lines were, after DNase treatment, submitted to reverse transcription with Superscript II (Invitrogen) using oligo dT as primer and the cDNA was used as template for PCR amplification with HLA-DRA primers. After 25, 28, 30 and 32 amplification cycles, 5 μl aliquots were collected for agarose gel electrophoresis. As endogenous control, a pair of primers for the ACTB (β-actin) mRNA was used. C: Control RT-PCR amplification using as template RNA (DNase treated) without prior reverse transcription.

Mentions: The identification of only 2% of the clones shared by both libraries strongly suggested that the cDNA subtraction was highly efficient. However, to confirm that this was indeed the case, we selected 7 genes for validation by Northern blots and RT-PCR in a panel of 6–8 melanoma cell lines that represent the three stages of tumor progression, including the cell lines used for the SSH libraries (Fig. 3). The genes selected for validation from the RGP library were DCN (represented by 8 clones), ALS2CR7 (10 clones) and MBOAT1 (3 clones). DCN encodes decorin, a secreted protein involved in cell growth regulation and apoptosis induction in tumors [43]. By Northern blot (Fig. 3A), we detected DCN mRNA only in the WM1552C cells and at very high levels, indicating that these libraries represent genes highly differentially expressed. ALS2CR7, a candidate gene of amyotrophic lateral sclerosis 2, encodes a putative protein kinase, based on Gene Ontology prediction, with no characterized function. As shown in Fig. 3B, although some signal for ALS2CR7 mRNA expression was detected in all eight cell lines analyzed, all four metastatic cell lines presented equivalently low signals and the highest levels were detected in WM1552C, confirming the differential expression of this gene identified in the RGP SSH library. The MBOAT1 gene encodes a hypothetical transmembrane protein containing an O-acyltransferase domain, also with no characterized function and as predicted by its occurrence in the RGP library, we confirmed that its mRNA expression is higher in WM1552C (Fig. 3C). On the other hand, when a fragment of a gene identified in both libraries, YWHAZ (14.3.3ζ) was used as probe for Northern blot hybridization (Fig. 3D), we detected average signals of similar intensity between the RGP WM1552C and the metastatic cell lines, although some variation in the expression of this gene can be noted among the cell lines analyzed.


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

Sousa JF, Espreafico EM - BMC Cancer (2008)

Validation by Northern blot and RT-PCR of the expression pattern of seven genes identified in the SSH libraries. Frames depict the names of cell lines used in the construction of the libraries. The inserts of cDNA clones corresponding to the genes DCN (decorin) (A), ALS2CR7 (B) and MBOAT1 (C) of the RGP library; YWHAZ (14-3-3 ξ) (D) identified in both libraries; and MITF (E) and PLP1 (F) from the Met library were isolated and used as probes for hybridization in Northern blots containing total RNA from the melanoma cell lines indicated above the panels – Blank lanes mean that the corresponding cell line was not included in the Northern blot, and were introduced to allow alignment among panels. Northern blots were prepared as described in Figure 1. HLA-DRA (G) identified in the Met library was validated by RT-PCR. For RT-PCR, total RNA samples (2 μg) from the indicated cell lines were, after DNase treatment, submitted to reverse transcription with Superscript II (Invitrogen) using oligo dT as primer and the cDNA was used as template for PCR amplification with HLA-DRA primers. After 25, 28, 30 and 32 amplification cycles, 5 μl aliquots were collected for agarose gel electrophoresis. As endogenous control, a pair of primers for the ACTB (β-actin) mRNA was used. C: Control RT-PCR amplification using as template RNA (DNase treated) without prior reverse transcription.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: Validation by Northern blot and RT-PCR of the expression pattern of seven genes identified in the SSH libraries. Frames depict the names of cell lines used in the construction of the libraries. The inserts of cDNA clones corresponding to the genes DCN (decorin) (A), ALS2CR7 (B) and MBOAT1 (C) of the RGP library; YWHAZ (14-3-3 ξ) (D) identified in both libraries; and MITF (E) and PLP1 (F) from the Met library were isolated and used as probes for hybridization in Northern blots containing total RNA from the melanoma cell lines indicated above the panels – Blank lanes mean that the corresponding cell line was not included in the Northern blot, and were introduced to allow alignment among panels. Northern blots were prepared as described in Figure 1. HLA-DRA (G) identified in the Met library was validated by RT-PCR. For RT-PCR, total RNA samples (2 μg) from the indicated cell lines were, after DNase treatment, submitted to reverse transcription with Superscript II (Invitrogen) using oligo dT as primer and the cDNA was used as template for PCR amplification with HLA-DRA primers. After 25, 28, 30 and 32 amplification cycles, 5 μl aliquots were collected for agarose gel electrophoresis. As endogenous control, a pair of primers for the ACTB (β-actin) mRNA was used. C: Control RT-PCR amplification using as template RNA (DNase treated) without prior reverse transcription.
Mentions: The identification of only 2% of the clones shared by both libraries strongly suggested that the cDNA subtraction was highly efficient. However, to confirm that this was indeed the case, we selected 7 genes for validation by Northern blots and RT-PCR in a panel of 6–8 melanoma cell lines that represent the three stages of tumor progression, including the cell lines used for the SSH libraries (Fig. 3). The genes selected for validation from the RGP library were DCN (represented by 8 clones), ALS2CR7 (10 clones) and MBOAT1 (3 clones). DCN encodes decorin, a secreted protein involved in cell growth regulation and apoptosis induction in tumors [43]. By Northern blot (Fig. 3A), we detected DCN mRNA only in the WM1552C cells and at very high levels, indicating that these libraries represent genes highly differentially expressed. ALS2CR7, a candidate gene of amyotrophic lateral sclerosis 2, encodes a putative protein kinase, based on Gene Ontology prediction, with no characterized function. As shown in Fig. 3B, although some signal for ALS2CR7 mRNA expression was detected in all eight cell lines analyzed, all four metastatic cell lines presented equivalently low signals and the highest levels were detected in WM1552C, confirming the differential expression of this gene identified in the RGP SSH library. The MBOAT1 gene encodes a hypothetical transmembrane protein containing an O-acyltransferase domain, also with no characterized function and as predicted by its occurrence in the RGP library, we confirmed that its mRNA expression is higher in WM1552C (Fig. 3C). On the other hand, when a fragment of a gene identified in both libraries, YWHAZ (14.3.3ζ) was used as probe for Northern blot hybridization (Fig. 3D), we detected average signals of similar intensity between the RGP WM1552C and the metastatic cell lines, although some variation in the expression of this gene can be noted among the cell lines analyzed.

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