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Establishment and characterization of HROC69 - a Crohn´s related colonic carcinoma cell line and its matched patient-derived xenograft.

Kuehn F, Mullins CS, Krohn M, Harnack C, Ramer R, Krämer OH, Klar E, Huehns M, Linnebacher M - Sci Rep (2016)

Bottom Line: They show strong invasive but in comparison weak migratory activity.The present work is the first description of patient-derived in vitro and in vivo models for CAC from a Crohn´s disease patient.They might be valuable tools for analysis of genetic and epigenetic alterations, biomarker identification, functional testing, including response prediction, and the development of specific therapeutical strategies.

View Article: PubMed Central - PubMed

Affiliation: University Medicine Rostock, Department of General-, Thoracic-, Vascular- and Transplantation Surgery, Rostock, Germany.

ABSTRACT
Colitis-associated colorectal cancer (CAC) seems to be a rather unique entity and differs in its genetic alterations, tumour formation capacities, and clinical features from sporadic colorectal carcinoma. Most descriptions about tumour biology of CAC refer to ulcerative colitis; data about Crohn´s colitis related carcinomas are scarce. The majority of patients with Crohn´s disease are under immunosuppression which generates a different environment for tumour growth. We first describe the clinical case of a fast growing CAC in a long-term immunosuppressed patient with Crohn´s disease and successful establishment and characterization of carcinoma cell lines along with their corresponding patient-derived xenograft. Subsequently, these tumor models were molecularly and functionally analysed. Beside numerous chromosomal alterations, mutations in TP53, APC, PTEN and SMAD3 were identified. The cell lines express numerous cancer testis antigens, surface molecules involved in immune evasion but low levels of HLA class I molecules. They show strong invasive but in comparison weak migratory activity. The present work is the first description of patient-derived in vitro and in vivo models for CAC from a Crohn´s disease patient. They might be valuable tools for analysis of genetic and epigenetic alterations, biomarker identification, functional testing, including response prediction, and the development of specific therapeutical strategies.

No MeSH data available.


Related in: MedlinePlus

Detection of proteins involved in tumour suppression, apoptosis and stress response by immune blot.Lysates from exponentially growing cultures of the patient-derived cell lines HROC69, HROC59 and HROC60 were subjected to Western blot analysis as indicated. Expression of p53 and its target genes p21, PIG3, BAX (positively regulated) and survivin (negatively regulated), HDAC1, HDAC2, and HSP90 as the loading control were determined with specific antibodies. We used 12% gels and performed the wet transfer procedure. All antibody incubations were done overnight at 4 °C. To ensure the correct molecular weight of the proteins that we detected, we used the molecular weight standard Fermentas #SM1811.
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f4: Detection of proteins involved in tumour suppression, apoptosis and stress response by immune blot.Lysates from exponentially growing cultures of the patient-derived cell lines HROC69, HROC59 and HROC60 were subjected to Western blot analysis as indicated. Expression of p53 and its target genes p21, PIG3, BAX (positively regulated) and survivin (negatively regulated), HDAC1, HDAC2, and HSP90 as the loading control were determined with specific antibodies. We used 12% gels and performed the wet transfer procedure. All antibody incubations were done overnight at 4 °C. To ensure the correct molecular weight of the proteins that we detected, we used the molecular weight standard Fermentas #SM1811.

Mentions: Our data show that while the tumour suppressor protein p53 is detectable in HROC69 cells, it occurs as a faster migrating band. The levels of this shorter p53 variant were expressed at levels comparable to wild-type p53 in HROC59 and to a lower extent than mutant p53 R273H in HROC60 cells (Fig. 4).


Establishment and characterization of HROC69 - a Crohn´s related colonic carcinoma cell line and its matched patient-derived xenograft.

Kuehn F, Mullins CS, Krohn M, Harnack C, Ramer R, Krämer OH, Klar E, Huehns M, Linnebacher M - Sci Rep (2016)

Detection of proteins involved in tumour suppression, apoptosis and stress response by immune blot.Lysates from exponentially growing cultures of the patient-derived cell lines HROC69, HROC59 and HROC60 were subjected to Western blot analysis as indicated. Expression of p53 and its target genes p21, PIG3, BAX (positively regulated) and survivin (negatively regulated), HDAC1, HDAC2, and HSP90 as the loading control were determined with specific antibodies. We used 12% gels and performed the wet transfer procedure. All antibody incubations were done overnight at 4 °C. To ensure the correct molecular weight of the proteins that we detected, we used the molecular weight standard Fermentas #SM1811.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834534&req=5

f4: Detection of proteins involved in tumour suppression, apoptosis and stress response by immune blot.Lysates from exponentially growing cultures of the patient-derived cell lines HROC69, HROC59 and HROC60 were subjected to Western blot analysis as indicated. Expression of p53 and its target genes p21, PIG3, BAX (positively regulated) and survivin (negatively regulated), HDAC1, HDAC2, and HSP90 as the loading control were determined with specific antibodies. We used 12% gels and performed the wet transfer procedure. All antibody incubations were done overnight at 4 °C. To ensure the correct molecular weight of the proteins that we detected, we used the molecular weight standard Fermentas #SM1811.
Mentions: Our data show that while the tumour suppressor protein p53 is detectable in HROC69 cells, it occurs as a faster migrating band. The levels of this shorter p53 variant were expressed at levels comparable to wild-type p53 in HROC59 and to a lower extent than mutant p53 R273H in HROC60 cells (Fig. 4).

Bottom Line: They show strong invasive but in comparison weak migratory activity.The present work is the first description of patient-derived in vitro and in vivo models for CAC from a Crohn´s disease patient.They might be valuable tools for analysis of genetic and epigenetic alterations, biomarker identification, functional testing, including response prediction, and the development of specific therapeutical strategies.

View Article: PubMed Central - PubMed

Affiliation: University Medicine Rostock, Department of General-, Thoracic-, Vascular- and Transplantation Surgery, Rostock, Germany.

ABSTRACT
Colitis-associated colorectal cancer (CAC) seems to be a rather unique entity and differs in its genetic alterations, tumour formation capacities, and clinical features from sporadic colorectal carcinoma. Most descriptions about tumour biology of CAC refer to ulcerative colitis; data about Crohn´s colitis related carcinomas are scarce. The majority of patients with Crohn´s disease are under immunosuppression which generates a different environment for tumour growth. We first describe the clinical case of a fast growing CAC in a long-term immunosuppressed patient with Crohn´s disease and successful establishment and characterization of carcinoma cell lines along with their corresponding patient-derived xenograft. Subsequently, these tumor models were molecularly and functionally analysed. Beside numerous chromosomal alterations, mutations in TP53, APC, PTEN and SMAD3 were identified. The cell lines express numerous cancer testis antigens, surface molecules involved in immune evasion but low levels of HLA class I molecules. They show strong invasive but in comparison weak migratory activity. The present work is the first description of patient-derived in vitro and in vivo models for CAC from a Crohn´s disease patient. They might be valuable tools for analysis of genetic and epigenetic alterations, biomarker identification, functional testing, including response prediction, and the development of specific therapeutical strategies.

No MeSH data available.


Related in: MedlinePlus