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Use of H19 Gene Regulatory Sequences in DNA-Based Therapy for Pancreatic Cancer.

Scaiewicz V, Sorin V, Fellig Y, Birman T, Mizrahi A, Galula J, Abu-Lail R, Shneider T, Ohana P, Buscail L, Hochberg A, Czerniak A - J Oncol (2010)

Bottom Line: In vitro experiments showed that the vector was effective in reducing Luciferase protein activity on pancreatic carcinoma cell lines.In addition, no visible metastases were found in the treated group of the orthotopic model.These results indicate that the treatment with the vector DTA-H19 might be a viable new therapeutic option for patients with unresectable pancreatic cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond Safra Campus, Givat Ram, Jerusalem 91904, Israel.

ABSTRACT
Pancreatic cancer is the eighth most common cause of death from cancer in the world, for which palliative treatments are not effective and frequently accompanied by severe side effects. We propose a DNA-based therapy for pancreatic cancer using a nonviral vector, expressing the diphtheria toxin A chain under the control of the H19 gene regulatory sequences. The H19 gene is an oncofetal RNA expressed during embryo development and in several types of cancer. We tested the expression of H19 gene in patients, and found that 65% of human pancreatic tumors analyzed showed moderated to strong expression of the gene. In vitro experiments showed that the vector was effective in reducing Luciferase protein activity on pancreatic carcinoma cell lines. In vivo experiment results revealed tumor growth arrest in different animal models for pancreatic cancer. Differences in tumor size between control and treated groups reached a 75% in the heterotopic model (P = .037) and 50% in the orthotopic model (P = .007). In addition, no visible metastases were found in the treated group of the orthotopic model. These results indicate that the treatment with the vector DTA-H19 might be a viable new therapeutic option for patients with unresectable pancreatic cancer.

No MeSH data available.


Related in: MedlinePlus

H19 RNA level in human and hamster pancreatic carcinoma cell lines. (a) Lane 1: 100 bp marker, Lane 2: CRL-1469 cells, Lane 3: PC.1-0 hamster cells. (b) Lane 1: 100 bp marker, Lanes 2–5: CRL-2547, CRL-1687, CRL-2119, and CRL-1997 human pancreatic carcinoma cells, respectively. (c) Lane 1: 100 bp marker; Lane 2: mouse control; Lane 3: PC.1-0 cells cultured under normoxia conditions; Lane 4: PC.1-0 cells cultured under hypoxia condition for 4 hours; Lane 5: tumors generated by PC.1-0 cells injection into nude mice back; Lane 6: negative control, no cDNA is present in the reaction mixture. The upper and the middle panels show the 228 bp and the 454 bp PCR products, respectively, obtained using two different primers (H19-mouse 228 and H19-mouse 454 as described in Supplementary data, Table A). The lower panel indicates the 400 bp β-actin internal control.
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fig2: H19 RNA level in human and hamster pancreatic carcinoma cell lines. (a) Lane 1: 100 bp marker, Lane 2: CRL-1469 cells, Lane 3: PC.1-0 hamster cells. (b) Lane 1: 100 bp marker, Lanes 2–5: CRL-2547, CRL-1687, CRL-2119, and CRL-1997 human pancreatic carcinoma cells, respectively. (c) Lane 1: 100 bp marker; Lane 2: mouse control; Lane 3: PC.1-0 cells cultured under normoxia conditions; Lane 4: PC.1-0 cells cultured under hypoxia condition for 4 hours; Lane 5: tumors generated by PC.1-0 cells injection into nude mice back; Lane 6: negative control, no cDNA is present in the reaction mixture. The upper and the middle panels show the 228 bp and the 454 bp PCR products, respectively, obtained using two different primers (H19-mouse 228 and H19-mouse 454 as described in Supplementary data, Table A). The lower panel indicates the 400 bp β-actin internal control.

Mentions: Three of the analyzed cell lines (CRL-2547, CRL-1687, and CRL-1469) showed high level of H19 expression (Figure 2(b), lanes 2 and 3 and Figure 2(a), lane 2, resp.), whereas the other two cell lines (CRL-2119 and CRL-1997) showed low levels of H19 expression (Figure 2(b), lanes 4 and 5, resp.). H19 expression was also analyzed on PC.1-0 cells, a hamster pancreatic carcinoma cell line. Since there is no report of the hamster's H19 gene sequence at the gene databases Ensembl and NCBI, primers designed originally for human and murine known H19 sequence were tested to determine H19 RNA level in hamster PC.1-0 cells. A faint band was visualized when primers designed originally for mouse H19 gene sequence were used (Figure 2(c), middle panel, lane 3). Previous studies showed that H19 gene expression may be induced by hypoxia and/or serum starvation conditions and that the H19 gene may play a role in tumorogenesis [17, 20]. Therefore, the level of H19 RNA in PC.1-0 was determined by RT-PCR extracted from cells cultured under hypoxia conditions for four hours, and also in total RNA extracted from tumors generated in athymic nude mice back by injecting 2*106 PC.1-0 cells (cells injected were cultured under normal conditions). When amplifying total RNA from cells cultured under hypoxia, a weak band was visualized using the set of primers H19_mouse (Figure 2(c), lane 4, middle panel; primers 454 bp Supplement Table A). On the other hand, when RNA extracted from the PC.1-0 developed tumors was analyzed, strong bands were visualized using the same set of primers (Figure 2(c), lane 5, upper and middle panel). These strong bands were purified and sequenced. BLAST analysis and sequences alignment showed high homology between the PCR products and the available sequences of mouse and human on gene databases (data not shown). Despite the high homology between the sequences, further analysis must be performed in order to assure that the sequence corresponds indeed to the hamster H19 RNA.


Use of H19 Gene Regulatory Sequences in DNA-Based Therapy for Pancreatic Cancer.

Scaiewicz V, Sorin V, Fellig Y, Birman T, Mizrahi A, Galula J, Abu-Lail R, Shneider T, Ohana P, Buscail L, Hochberg A, Czerniak A - J Oncol (2010)

H19 RNA level in human and hamster pancreatic carcinoma cell lines. (a) Lane 1: 100 bp marker, Lane 2: CRL-1469 cells, Lane 3: PC.1-0 hamster cells. (b) Lane 1: 100 bp marker, Lanes 2–5: CRL-2547, CRL-1687, CRL-2119, and CRL-1997 human pancreatic carcinoma cells, respectively. (c) Lane 1: 100 bp marker; Lane 2: mouse control; Lane 3: PC.1-0 cells cultured under normoxia conditions; Lane 4: PC.1-0 cells cultured under hypoxia condition for 4 hours; Lane 5: tumors generated by PC.1-0 cells injection into nude mice back; Lane 6: negative control, no cDNA is present in the reaction mixture. The upper and the middle panels show the 228 bp and the 454 bp PCR products, respectively, obtained using two different primers (H19-mouse 228 and H19-mouse 454 as described in Supplementary data, Table A). The lower panel indicates the 400 bp β-actin internal control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig2: H19 RNA level in human and hamster pancreatic carcinoma cell lines. (a) Lane 1: 100 bp marker, Lane 2: CRL-1469 cells, Lane 3: PC.1-0 hamster cells. (b) Lane 1: 100 bp marker, Lanes 2–5: CRL-2547, CRL-1687, CRL-2119, and CRL-1997 human pancreatic carcinoma cells, respectively. (c) Lane 1: 100 bp marker; Lane 2: mouse control; Lane 3: PC.1-0 cells cultured under normoxia conditions; Lane 4: PC.1-0 cells cultured under hypoxia condition for 4 hours; Lane 5: tumors generated by PC.1-0 cells injection into nude mice back; Lane 6: negative control, no cDNA is present in the reaction mixture. The upper and the middle panels show the 228 bp and the 454 bp PCR products, respectively, obtained using two different primers (H19-mouse 228 and H19-mouse 454 as described in Supplementary data, Table A). The lower panel indicates the 400 bp β-actin internal control.
Mentions: Three of the analyzed cell lines (CRL-2547, CRL-1687, and CRL-1469) showed high level of H19 expression (Figure 2(b), lanes 2 and 3 and Figure 2(a), lane 2, resp.), whereas the other two cell lines (CRL-2119 and CRL-1997) showed low levels of H19 expression (Figure 2(b), lanes 4 and 5, resp.). H19 expression was also analyzed on PC.1-0 cells, a hamster pancreatic carcinoma cell line. Since there is no report of the hamster's H19 gene sequence at the gene databases Ensembl and NCBI, primers designed originally for human and murine known H19 sequence were tested to determine H19 RNA level in hamster PC.1-0 cells. A faint band was visualized when primers designed originally for mouse H19 gene sequence were used (Figure 2(c), middle panel, lane 3). Previous studies showed that H19 gene expression may be induced by hypoxia and/or serum starvation conditions and that the H19 gene may play a role in tumorogenesis [17, 20]. Therefore, the level of H19 RNA in PC.1-0 was determined by RT-PCR extracted from cells cultured under hypoxia conditions for four hours, and also in total RNA extracted from tumors generated in athymic nude mice back by injecting 2*106 PC.1-0 cells (cells injected were cultured under normal conditions). When amplifying total RNA from cells cultured under hypoxia, a weak band was visualized using the set of primers H19_mouse (Figure 2(c), lane 4, middle panel; primers 454 bp Supplement Table A). On the other hand, when RNA extracted from the PC.1-0 developed tumors was analyzed, strong bands were visualized using the same set of primers (Figure 2(c), lane 5, upper and middle panel). These strong bands were purified and sequenced. BLAST analysis and sequences alignment showed high homology between the PCR products and the available sequences of mouse and human on gene databases (data not shown). Despite the high homology between the sequences, further analysis must be performed in order to assure that the sequence corresponds indeed to the hamster H19 RNA.

Bottom Line: In vitro experiments showed that the vector was effective in reducing Luciferase protein activity on pancreatic carcinoma cell lines.In addition, no visible metastases were found in the treated group of the orthotopic model.These results indicate that the treatment with the vector DTA-H19 might be a viable new therapeutic option for patients with unresectable pancreatic cancer.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Chemistry, Institute of Life Sciences, The Hebrew University of Jerusalem, Edmond Safra Campus, Givat Ram, Jerusalem 91904, Israel.

ABSTRACT
Pancreatic cancer is the eighth most common cause of death from cancer in the world, for which palliative treatments are not effective and frequently accompanied by severe side effects. We propose a DNA-based therapy for pancreatic cancer using a nonviral vector, expressing the diphtheria toxin A chain under the control of the H19 gene regulatory sequences. The H19 gene is an oncofetal RNA expressed during embryo development and in several types of cancer. We tested the expression of H19 gene in patients, and found that 65% of human pancreatic tumors analyzed showed moderated to strong expression of the gene. In vitro experiments showed that the vector was effective in reducing Luciferase protein activity on pancreatic carcinoma cell lines. In vivo experiment results revealed tumor growth arrest in different animal models for pancreatic cancer. Differences in tumor size between control and treated groups reached a 75% in the heterotopic model (P = .037) and 50% in the orthotopic model (P = .007). In addition, no visible metastases were found in the treated group of the orthotopic model. These results indicate that the treatment with the vector DTA-H19 might be a viable new therapeutic option for patients with unresectable pancreatic cancer.

No MeSH data available.


Related in: MedlinePlus