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Cyclin-Dependent Kinase 5 (CDK5) Controls Melanoma Cell Motility, Invasiveness, and Metastatic Spread-Identification of a Promising Novel therapeutic target.

Bisht S, Nolting J, Schütte U, Haarmann J, Jain P, Shah D, Brossart P, Flaherty P, Feldmann G - Transl Oncol (2015)

Bottom Line: In vivo, CDK5 knockdown inhibited growth of orthotopic xenografts as well as formation of lung and liver colonies in xenogenic injection models mimicking systemic metastases.CDK5 knockdown was accompanied by dephosphorylation and overexpression of caldesmon, and concomitant caldesmon knockdown rescued cell motility and proinvasive phenotype.Finally, it was found that pharmacological inhibition of CDK5 activity by means of roscovitine as well as by a novel small molecule CDK5-inhibitor, N-(5-isopropylthiazol-2-yl)-3-phenylpropanamide, similarly caused marked inhibition of invasion/migration, colony formation, and anchorage-independent growth of melanoma cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine 3, Center of Integrated Oncology (CIO) Cologne-Bonn, University Hospital of Bonn, Germany.

No MeSH data available.


Related in: MedlinePlus

shRNA-mediated knockdown of endogenous CDK5 inhibits anchorage-independent growth of melanoma cells. An inducible pTRIPZ-Tet-On expression vector system containing CDK5-specific shRNA was used to knock down CDK5 expression in SKMel28 human malignant melanoma cells. (A) Transduction efficiency was confirmed by red fluorescent protein expression as visualized using fluorescence microscopy. (B) Successful inducible CDK5 knockdown was checked using Western blot analysis (upper panel) and quantitative real-time RT-PCR analysis (lower panel) both in the absence and in the presence of doxycycline, respectively. (C) Inducible knockdown of CDK5 did not show any significant effect on the net cell growth of SKMel28 cells as assessed using MTS assay. The figure shows results of two different pairs of mass clones (designated “SKMel28-F10” and SKMel28-B7” with their respective mock controls) both in the presence (= induced state) and in the absence (= uninduced state) of doxycycline. (D) In contrast, colony formation and anchorage-independent growth in soft agar were significantly reduced in both SKMel28 mass clones when induced by doxycycline as compared with uninduced or mock-transduced controls. Colony counts are plotted as means and SD from three independent experiments; *P < .05.
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f0010: shRNA-mediated knockdown of endogenous CDK5 inhibits anchorage-independent growth of melanoma cells. An inducible pTRIPZ-Tet-On expression vector system containing CDK5-specific shRNA was used to knock down CDK5 expression in SKMel28 human malignant melanoma cells. (A) Transduction efficiency was confirmed by red fluorescent protein expression as visualized using fluorescence microscopy. (B) Successful inducible CDK5 knockdown was checked using Western blot analysis (upper panel) and quantitative real-time RT-PCR analysis (lower panel) both in the absence and in the presence of doxycycline, respectively. (C) Inducible knockdown of CDK5 did not show any significant effect on the net cell growth of SKMel28 cells as assessed using MTS assay. The figure shows results of two different pairs of mass clones (designated “SKMel28-F10” and SKMel28-B7” with their respective mock controls) both in the presence (= induced state) and in the absence (= uninduced state) of doxycycline. (D) In contrast, colony formation and anchorage-independent growth in soft agar were significantly reduced in both SKMel28 mass clones when induced by doxycycline as compared with uninduced or mock-transduced controls. Colony counts are plotted as means and SD from three independent experiments; *P < .05.

Mentions: To study the role of CDK5 in melanoma progression and metastasis, CDK5 function was abrogated using short hairpin RNA interference in melanoma cells. An inducible lentiviral pTRIPZ-Tet-On vector system was used for this purpose in which expression of shRNA is induced in the presence of doxycycline; red fluorescent protein served as control of transduction efficiency. SKMel28 melanoma cells were stably transduced with one of two independent CDK5-specific shRNA clones (designated “SKMel-F10” or “SKMel-B7,” respectively) or with empty pTRIPZ vector as mock control. Transduction efficiency and specificity of doxycycline induction were tested using fluorescence microscopy (Figure 2A). Western blot analysis and quantitative real-time RT-PCR showed successful and reproducible knockdown of CDK5 upon addition of doxycycline in SKMel-F10 and SKMel-B7 mass clones at both the protein and mRNA level, but not in mock-transduced vector-only controls or in the absence of doxycycline (Figure 2B).


Cyclin-Dependent Kinase 5 (CDK5) Controls Melanoma Cell Motility, Invasiveness, and Metastatic Spread-Identification of a Promising Novel therapeutic target.

Bisht S, Nolting J, Schütte U, Haarmann J, Jain P, Shah D, Brossart P, Flaherty P, Feldmann G - Transl Oncol (2015)

shRNA-mediated knockdown of endogenous CDK5 inhibits anchorage-independent growth of melanoma cells. An inducible pTRIPZ-Tet-On expression vector system containing CDK5-specific shRNA was used to knock down CDK5 expression in SKMel28 human malignant melanoma cells. (A) Transduction efficiency was confirmed by red fluorescent protein expression as visualized using fluorescence microscopy. (B) Successful inducible CDK5 knockdown was checked using Western blot analysis (upper panel) and quantitative real-time RT-PCR analysis (lower panel) both in the absence and in the presence of doxycycline, respectively. (C) Inducible knockdown of CDK5 did not show any significant effect on the net cell growth of SKMel28 cells as assessed using MTS assay. The figure shows results of two different pairs of mass clones (designated “SKMel28-F10” and SKMel28-B7” with their respective mock controls) both in the presence (= induced state) and in the absence (= uninduced state) of doxycycline. (D) In contrast, colony formation and anchorage-independent growth in soft agar were significantly reduced in both SKMel28 mass clones when induced by doxycycline as compared with uninduced or mock-transduced controls. Colony counts are plotted as means and SD from three independent experiments; *P < .05.
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f0010: shRNA-mediated knockdown of endogenous CDK5 inhibits anchorage-independent growth of melanoma cells. An inducible pTRIPZ-Tet-On expression vector system containing CDK5-specific shRNA was used to knock down CDK5 expression in SKMel28 human malignant melanoma cells. (A) Transduction efficiency was confirmed by red fluorescent protein expression as visualized using fluorescence microscopy. (B) Successful inducible CDK5 knockdown was checked using Western blot analysis (upper panel) and quantitative real-time RT-PCR analysis (lower panel) both in the absence and in the presence of doxycycline, respectively. (C) Inducible knockdown of CDK5 did not show any significant effect on the net cell growth of SKMel28 cells as assessed using MTS assay. The figure shows results of two different pairs of mass clones (designated “SKMel28-F10” and SKMel28-B7” with their respective mock controls) both in the presence (= induced state) and in the absence (= uninduced state) of doxycycline. (D) In contrast, colony formation and anchorage-independent growth in soft agar were significantly reduced in both SKMel28 mass clones when induced by doxycycline as compared with uninduced or mock-transduced controls. Colony counts are plotted as means and SD from three independent experiments; *P < .05.
Mentions: To study the role of CDK5 in melanoma progression and metastasis, CDK5 function was abrogated using short hairpin RNA interference in melanoma cells. An inducible lentiviral pTRIPZ-Tet-On vector system was used for this purpose in which expression of shRNA is induced in the presence of doxycycline; red fluorescent protein served as control of transduction efficiency. SKMel28 melanoma cells were stably transduced with one of two independent CDK5-specific shRNA clones (designated “SKMel-F10” or “SKMel-B7,” respectively) or with empty pTRIPZ vector as mock control. Transduction efficiency and specificity of doxycycline induction were tested using fluorescence microscopy (Figure 2A). Western blot analysis and quantitative real-time RT-PCR showed successful and reproducible knockdown of CDK5 upon addition of doxycycline in SKMel-F10 and SKMel-B7 mass clones at both the protein and mRNA level, but not in mock-transduced vector-only controls or in the absence of doxycycline (Figure 2B).

Bottom Line: In vivo, CDK5 knockdown inhibited growth of orthotopic xenografts as well as formation of lung and liver colonies in xenogenic injection models mimicking systemic metastases.CDK5 knockdown was accompanied by dephosphorylation and overexpression of caldesmon, and concomitant caldesmon knockdown rescued cell motility and proinvasive phenotype.Finally, it was found that pharmacological inhibition of CDK5 activity by means of roscovitine as well as by a novel small molecule CDK5-inhibitor, N-(5-isopropylthiazol-2-yl)-3-phenylpropanamide, similarly caused marked inhibition of invasion/migration, colony formation, and anchorage-independent growth of melanoma cells.

View Article: PubMed Central - PubMed

Affiliation: Department of Internal Medicine 3, Center of Integrated Oncology (CIO) Cologne-Bonn, University Hospital of Bonn, Germany.

No MeSH data available.


Related in: MedlinePlus