Fluorescent peptide biosensor for monitoring CDK4/cyclin D kinase activity in melanoma cell extracts, mouse xenografts and skin biopsies

Prével, Camille; Pellerano, Morgan; González‐Vera, Juan A.; Henri, Pauline; Meunier, Laurent; Vollaire, Julien; Josserand, Véronique; Morris, May

doi:10.1016/j.bios.2016.04.050

Cited by 18 publications

(42 citation statements)

References 34 publications

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“…CDKACT biosensors are environmentally sensitive fluorescent peptide biosensors that undergo fluorescence enhancement upon phosphorylation by CDK or cyclins. TAMRA‐labelled CDKACT4 is derived from the retinoblastoma tumour suppressor protein (Rb) substrate and reports on CDK4 and cyclin D activity, while Cy3‐labelled CDKACT1 is derived from histone H1 and reports on CDKs in a broader fashion . Fluorescence titration assays were performed as described previously in 96‐well plates in a thermostat chamber (Clariostar; BMG Labtech, Ortenberg, Germany) at 30 °C.…”

Section: Methodsmentioning

confidence: 99%

“…In this report, we implemented fluorescent biosensors, which have been validated and described elsewhere, to monitor CDK4 and CDK2 activity in human epidermis obtained from normal‐appearing perilesional and lesional psoriatic skin. By using confocal and Western blot analysis, we also evaluated the expression of CDK4 and CDK2, CycD1 and CycE, and the CDK structural inhibitors p16 INK4A (p16), p21 Cip1 (p21) and p27 Kip1 (p27).…”

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confidence: 99%

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Psoriatic epidermis is associated with upregulation of CDK 2 and inhibition of CDK 4 activity

et al. 2019

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Summary Background The cyclin‐dependent kinases (CDKs) CDK2 and CDK4 are involved in regulation of cell‐cycle progression, and psoriasis is characterized by hyperproliferation of basal epidermal cells. CDK inhibitory proteins (CKIs) such as p16INK4A (p16) bind CDK4/6 kinases and prevent their interaction with D‐type cyclins. CKIs such as p21Cip1 (p21) and p27Kip1 (p27) associate with CDK–cyclin complexes and prevent their activation. Objectives To gain insight into the molecular implication of CDK2 and CDK4 kinases in psoriasis, we sought to characterize expression of these kinases and associated cyclins, as well as of CKIs, and addressed the status of CDK2 and CDK4 activity in human psoriatic epidermis. Methods A cohort of 24 patients with psoriasis participated in the study. Biopsies were removed from a chronic plaque and from nonlesional skin. CDK2, CDK4, cyclin D1, cyclin E and CKI protein expression was assessed by immunoblotting, immunohistochemistry and immunofluorescence. CDK4 and CDK2 mRNA expression was determined by real‐time polymerase chain reaction. Specific kinase activities of CDK2 and CDK4 were evaluated using fluorescent peptide biosensors. Results CDK2–cyclin E expression and activity were significantly increased in psoriatic epidermis compared with uninvolved adjacent skin. In contrast, CDK4–cyclin D1 activity was inhibited, although its expression was increased in psoriatic epidermis and its transcription slightly inhibited. p27 expression was reduced, while p16 and p21 expression was induced in psoriatic epidermis. Conclusions Epidermal CDK2 activity is increased in psoriatic epidermis while CDK4 activity is completely inhibited. These alterations are not associated with changes in CDK transcription and instead involve post‐translational control mediated by decreased expression of p27 and p16 overexpression, respectively. What's already known about this topic? Cyclin‐dependent kinases (CDKs) are involved in cell‐cycle progression. The levels of cyclin partners and CDK inhibitors regulate their activity. Psoriasis is a chronic T‐cell‐driven inflammatory skin disease characterized by hyperproliferation of basal epidermal cells. What does this study add? Thanks to fluorescent peptide biosensors, this study demonstrates that epidermal CDK2 activity is increased in psoriatic epidermis while CDK4 activity is completely inhibited. These alterations involve post‐translational control mediated by decreased expression of p27, and p16 overexpression, respectively. What is the translational message? CDK2 and CDK4 are involved in regulation of cell‐cycle progression, and psoriasis is characterized by hyperproliferation of basal epidermal cells. Epidermal CDK2 activity is increased in psoriatic epidermis while CDK4 activity is completely inhibited. These alterations are not associated with changes in CDK transcription and instead involve post‐translational control mediated by decreased expression of p27 and p16 overexpression, respectively. Pharmacological modulation of CDK2 and CDK4 may c...

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Section: Methodsmentioning

confidence: 99%

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confidence: 99%

Psoriatic epidermis is associated with upregulation of CDK 2 and inhibition of CDK 4 activity

et al. 2019

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“… 160 CDK4/cyclin D kinase hyperactivation, associated with the mutation of CDK4 , amplification of cyclin D , or complete deletion of p16INK4a , leads to an increased risk of developing melanomas. 161 …”

Section: Targeted Therapymentioning

confidence: 99%

Melanoma treatment in review

Domingues

Lopes

Soares

et al. 2018

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531

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Melanoma represents the most aggressive and the deadliest form of skin cancer. Current therapeutic approaches include surgical resection, chemotherapy, photodynamic therapy, immunotherapy, biochemotherapy, and targeted therapy. The therapeutic strategy can include single agents or combined therapies, depending on the patient’s health, stage, and location of the tumor. The efficiency of these treatments can be decreased due to the development of diverse resistance mechanisms. New therapeutic targets have emerged from studies of the genetic profile of melanocytes and from the identification of molecular factors involved in the pathogenesis of the malignant transformation. In this review, we aim to survey therapies approved and under evaluation for melanoma treatment and relevant research on the molecular mechanisms underlying melanomagenesis.

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“…Eltayib et al have reported minimally invasive monitoring MNAs for monitoring lithium. (Eltayib, 2016) Similar experiment in Morris's lab, show the potential of MNAs to access intracellular CDK4 thanks to specific peptide substrates (Prével, 2016), in both cultured cells and frozen skin samples onto which they were applied. Western blot analyses reveal that CDK4 can be found on the microneedle arrays following insertion into A375 melanoma cells grown on plastic dishes.…”

Section: Insert Figure 3 Herementioning

confidence: 76%

Microneedle Array‐Based Platforms for Future Theranostic Applications

et al. 2019

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Theranostics involves finding the biomarkers of a disease, fighting them through site specific drug delivery and following them for prognosis of the disease. Microneedle array technology has been used for drug delivery and extended for continuous monitoring of analytes present in the skin compartment. We envisage the use of microneedle arrays for future theranostic applications. The potential of combining microneedle array‐based drug delivery and diagnostics as part of closed‐loop control system for the management of diseases and delivery of precision drugs in individual patients is reported in this paper.

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Fluorescent peptide biosensor for monitoring CDK4/cyclin D kinase activity in melanoma cell extracts, mouse xenografts and skin biopsies

Cited by 18 publications

References 34 publications

Psoriatic epidermis is associated with upregulation of CDK 2 and inhibition of CDK 4 activity

Psoriatic epidermis is associated with upregulation of CDK 2 and inhibition of CDK 4 activity

Melanoma treatment in review

Microneedle Array‐Based Platforms for Future Theranostic Applications

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