23 research outputs found
Co-activation of hedgehog and AKT pathways promote tumorigenesis in zebrafish
The zebrafish has become an important model for cancer research. Several cancer models have been established by transgenic expression of human or mouse oncogenes in zebrafish. Since it is amenable to efficient transgenesis, zebrafish have immense potential to be used for studying interaction of oncogenes and pathways at the organismal level. Using the Gal4VP16-UAS binary transgenic expression approach, we established stable transgenic lines expressing an EGFP fusion protein of an activated zebrafish Smoothened (Smoa1-EGFP). Expression of the zebrafish Smoa1-EGFP itself did not lead to tumor formation either in founder fish or subsequent generations, however, co-expressing a constitutively active human AKT1 resulted in several tumor types, including spindle cell sarcoma, rhabdomyoma, ocular melanoma, astrocytoma, and myoxma. All tumor types showed GFP expression and increased Patched 1 levels, suggesting involvement of zebrafish Smoa1 in tumorigenesis. Immunofluorescence studies showed that tumors also expressed elevated levels of phosphorylated AKT, indicating activation of the PI3K-AKT pathway. These results suggest that co-activation of the hedgehog and AKT pathways promote tumorigenesis, and that the binary transgenic approach is a useful tool for studying interaction of oncogenes and oncogenic pathways in zebrafish
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Co-activation of hedgehog and AKT pathways promote tumorigenesis in zebrafish
The zebrafish has become an important model for cancer research. Several cancer models have
been established by transgenic expression of human or mouse oncogenes in zebrafish. Since it is
amenable to efficient transgenesis, zebrafish have immense potential to be used for studying
interaction of oncogenes and pathways at the organismal level. Using the Gal4VP16-UAS binary
transgenic expression approach, we established stable transgenic lines expressing an EGFP fusion
protein of an activated zebrafish Smoothened (Smoa1-EGFP). Expression of the zebrafish Smoa1-EGFP itself did not lead to tumor formation either in founder fish or subsequent generations,
however, co-expressing a constitutively active human AKT1 resulted in several tumor types,
including spindle cell sarcoma, rhabdomyoma, ocular melanoma, astrocytoma, and myoxma. All
tumor types showed GFP expression and increased Patched 1 levels, suggesting involvement of
zebrafish Smoa1 in tumorigenesis. Immunofluorescence studies showed that tumors also expressed
elevated levels of phosphorylated AKT, indicating activation of the PI3K-AKT pathway. These
results suggest that co-activation of the hedgehog and AKT pathways promote tumorigenesis, and
that the binary transgenic approach is a useful tool for studying interaction of oncogenes and
oncogenic pathways in zebrafish
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Oncogenic KRAS promotes malignant brain tumors in zebrafish
BACKGROUND: Zebrafish have been used as a vertebrate model to study human cancers such as melanoma,
rhabdomyosarcoma, liver cancer, and leukemia as well as for high-throughput screening of small molecules of
therapeutic value. However, they are just emerging as a model for human brain tumors, which are among the most
devastating and difficult to treat. In this study, we evaluated zebrafish as a brain tumor model by overexpressing a
human version of oncogenic KRAS (KRAS[superscript G12V]).
METHODS: Using zebrafish cytokeratin 5 (krt5) and glial fibrillary acidic protein (gfap) gene promoters, we activated
Ras signaling in the zebrafish central nervous system (CNS) through transient and stable transgenic overexpression.
Immunohistochemical analyses were performed to identify activated pathways in the resulting brain tumors. The
effects of the MEK inhibitor U0126 on oncogenic KRAS were evaluated.
RESULTS: We demonstrated that transient transgenic expression of KRAS[superscript G12V] in putative neural stem and/or progenitor cells induced brain tumorigenesis. When expressed under the control of the krt5 gene promoter, KRAS[superscript G12V] induced brain tumors in ventricular zones (VZ) at low frequency. The majority of other tumors were composed mostly of spindle and epithelioid cells, reminiscent of malignant peripheral nerve sheath tumors (MPNSTs). In contrast, when expressed under the control of the gfap gene promoter, KRAS[superscript G12V] induced brain tumors in both VZs and brain parenchyma at higher frequency. Immunohistochemical analyses indicated prominent activation of the canonical RAS-RAF-ERK pathway, variable activation of the mTOR pathway, but no activation of the PI3K-AKT pathway. In a krt5-derived stable and inducible transgenic line, expression of oncogenic KRAS resulted in skin hyperplasia, and the MEK inhibitor U0126 effectively suppressed this pro-proliferative effects. In a gfap-derived stable and inducible line, expression of oncogenic KRAS led to significantly increased mitotic index in the spinal cord.
CONCLUSIONS: Our studies demonstrate that zebrafish could be explored to study cellular origins and molecular
mechanisms of brain tumorigenesis and could also be used as a platform for studying human oncogene function and
for discovering oncogenic RAS inhibitors.Keywords: gfap, krt5, Oncogenic KRAS (KRAS[superscript G12V]), Zebrafish, Drug screening, Brain tumor
CHIMERIC GENE CONSTRUCTS FOR GENERATION OF FLUORESCENT TRANSGENIC ORNAMENTAL FISH
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Sale of transgenic fish that express gene encoding fluorescent protein
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SpitsbergenJanMicrobiologyCo-ActivationHedgehogAKT.pdf
The zebrafish has become an important model for cancer research. Several cancer models have
been established by transgenic expression of human or mouse oncogenes in zebrafish. Since it is
amenable to efficient transgenesis, zebrafish have immense potential to be used for studying
interaction of oncogenes and pathways at the organismal level. Using the Gal4VP16-UAS binary
transgenic expression approach, we established stable transgenic lines expressing an EGFP fusion
protein of an activated zebrafish Smoothened (Smoa1-EGFP). Expression of the zebrafish Smoa1-EGFP itself did not lead to tumor formation either in founder fish or subsequent generations,
however, co-expressing a constitutively active human AKT1 resulted in several tumor types,
including spindle cell sarcoma, rhabdomyoma, ocular melanoma, astrocytoma, and myoxma. All
tumor types showed GFP expression and increased Patched 1 levels, suggesting involvement of
zebrafish Smoa1 in tumorigenesis. Immunofluorescence studies showed that tumors also expressed
elevated levels of phosphorylated AKT, indicating activation of the PI3K-AKT pathway. These
results suggest that co-activation of the hedgehog and AKT pathways promote tumorigenesis, and
that the binary transgenic approach is a useful tool for studying interaction of oncogenes and
oncogenic pathways in zebrafish
Chimeric gene constructs for generation of fluorescent transgenic ornamental fish
US7135613Granted Paten
Chimeric gene constructs for generation of fluorescent transgenic ornamental fish
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SpitsbergenJanMicrobiologyCo-ActivationHedgehogAKT(AdditionalFile2).jpg
The zebrafish has become an important model for cancer research. Several cancer models have
been established by transgenic expression of human or mouse oncogenes in zebrafish. Since it is
amenable to efficient transgenesis, zebrafish have immense potential to be used for studying
interaction of oncogenes and pathways at the organismal level. Using the Gal4VP16-UAS binary
transgenic expression approach, we established stable transgenic lines expressing an EGFP fusion
protein of an activated zebrafish Smoothened (Smoa1-EGFP). Expression of the zebrafish Smoa1-EGFP itself did not lead to tumor formation either in founder fish or subsequent generations,
however, co-expressing a constitutively active human AKT1 resulted in several tumor types,
including spindle cell sarcoma, rhabdomyoma, ocular melanoma, astrocytoma, and myoxma. All
tumor types showed GFP expression and increased Patched 1 levels, suggesting involvement of
zebrafish Smoa1 in tumorigenesis. Immunofluorescence studies showed that tumors also expressed
elevated levels of phosphorylated AKT, indicating activation of the PI3K-AKT pathway. These
results suggest that co-activation of the hedgehog and AKT pathways promote tumorigenesis, and
that the binary transgenic approach is a useful tool for studying interaction of oncogenes and
oncogenic pathways in zebrafish