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Concerted functions of HDAC1 and microRNA-574-5p repress alternatively spliced ceramide synthase 1 expression in human cancer cells.

Meyers-Needham M, Ponnusamy S, Gencer S, Jiang W, Thomas RJ, Senkal CE, Ogretmen B - EMBO Mol Med (2011)

Bottom Line: Moreover, an alternatively spliced variant CerS1 mRNA (CerS1-2) was detected mainly in cancer cells or primary tumour tissues compared to controls, which was targeted by miR-574-5p for degradation.Interference with HDAC1 and miR-574-5p reconstituted CerS1-2 expression and C(18) -ceramide generation in multiple human cancer cell lines, which subsequently inhibited proliferation and anchorage-independent growth.Accordingly, knockdown of CerS1 partially protected cancer cells from MS-275/miR-574-5p siRNA-mediated growth inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, SC, USA.

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Related in: MedlinePlus

Regulation of CerS1 versus CerS6 mRNA and promoter activities in HNSCC versus keratinocytesA-B.CerS1 mRNA was detected by Q-PCR in primary NHEK (A) or in HPV E6/E7-immortalized skin keratinocytes (B), compared to UM-SCC-1 and UM-SCC-22A cells by Q-PCR.C. Ceramide measurements were performed in immortalized skin keratinocytes compared to UM-SCC-1 and UM-SCC2A cells using LC/MS/MS. Data were normalized to Pi.D-E.CerS1 (D) and CerS6 (E) promoter activities were measured using luminometry, normalizing transfection efficiency to β-gal expression using spectrometry. Transcription factor binding sites were predicted using the TFBind software. Samples were run in duplicates at least three-independent times. The error bars represent the standard deviations.
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fig01: Regulation of CerS1 versus CerS6 mRNA and promoter activities in HNSCC versus keratinocytesA-B.CerS1 mRNA was detected by Q-PCR in primary NHEK (A) or in HPV E6/E7-immortalized skin keratinocytes (B), compared to UM-SCC-1 and UM-SCC-22A cells by Q-PCR.C. Ceramide measurements were performed in immortalized skin keratinocytes compared to UM-SCC-1 and UM-SCC2A cells using LC/MS/MS. Data were normalized to Pi.D-E.CerS1 (D) and CerS6 (E) promoter activities were measured using luminometry, normalizing transfection efficiency to β-gal expression using spectrometry. Transcription factor binding sites were predicted using the TFBind software. Samples were run in duplicates at least three-independent times. The error bars represent the standard deviations.

Mentions: To determine whether down-regulation of CerS1 mRNA in the majority of HNSCC tumour tissues compared to their adjacent normal head and neck tissues (Karahatay et al, 2007; Koybasi et al, 2004) is also consistent in cell culture conditions, we measured CerS1 and CerS6 mRNA in multiple human cancer cells (UM-SCC-22A, UM-SCC-14A and UM-SCC-1) compared to non-cancerous keratinocytes using quantitative-polymerase chain reaction (Q-PCR). Consistent with HNSCC primary tumours, CerS1 was down-regulated about 10–20-fold in HNSCC cell lines compared to normal human epidermal primary keratinocytes (NHEK) or HPV-E6/E7-immortalized human keratinocytes controls (Fig 1A and B). CerS6 mRNA was similar in HNSCC cell lines and immortalized keratinocytes (Fig 1B). Down-regulation of CerS1 was also associated with lower C18-ceramide, measured with liquid chromatography/mass spectrometry (LC/MS/MS), in UM-SCC-1 and -22A (Fig 1C) compared to immortalized keratinocytes (about 90 and 50%, respectively). Thus, these studies indicate that CerS1/C18-ceramide, but not CerS6/C16-ceramide is down-regulated in HNSCC cancer cell lines compared to non-cancerous keratinocytes, consistent with decreased CerS1/C18-ceramide in HNSCC tumour compared to non-cancerous head and neck tissues (Karahatay et al, 2007; Koybasi et al, 2004).


Concerted functions of HDAC1 and microRNA-574-5p repress alternatively spliced ceramide synthase 1 expression in human cancer cells.

Meyers-Needham M, Ponnusamy S, Gencer S, Jiang W, Thomas RJ, Senkal CE, Ogretmen B - EMBO Mol Med (2011)

Regulation of CerS1 versus CerS6 mRNA and promoter activities in HNSCC versus keratinocytesA-B.CerS1 mRNA was detected by Q-PCR in primary NHEK (A) or in HPV E6/E7-immortalized skin keratinocytes (B), compared to UM-SCC-1 and UM-SCC-22A cells by Q-PCR.C. Ceramide measurements were performed in immortalized skin keratinocytes compared to UM-SCC-1 and UM-SCC2A cells using LC/MS/MS. Data were normalized to Pi.D-E.CerS1 (D) and CerS6 (E) promoter activities were measured using luminometry, normalizing transfection efficiency to β-gal expression using spectrometry. Transcription factor binding sites were predicted using the TFBind software. Samples were run in duplicates at least three-independent times. The error bars represent the standard deviations.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3376837&req=5

fig01: Regulation of CerS1 versus CerS6 mRNA and promoter activities in HNSCC versus keratinocytesA-B.CerS1 mRNA was detected by Q-PCR in primary NHEK (A) or in HPV E6/E7-immortalized skin keratinocytes (B), compared to UM-SCC-1 and UM-SCC-22A cells by Q-PCR.C. Ceramide measurements were performed in immortalized skin keratinocytes compared to UM-SCC-1 and UM-SCC2A cells using LC/MS/MS. Data were normalized to Pi.D-E.CerS1 (D) and CerS6 (E) promoter activities were measured using luminometry, normalizing transfection efficiency to β-gal expression using spectrometry. Transcription factor binding sites were predicted using the TFBind software. Samples were run in duplicates at least three-independent times. The error bars represent the standard deviations.
Mentions: To determine whether down-regulation of CerS1 mRNA in the majority of HNSCC tumour tissues compared to their adjacent normal head and neck tissues (Karahatay et al, 2007; Koybasi et al, 2004) is also consistent in cell culture conditions, we measured CerS1 and CerS6 mRNA in multiple human cancer cells (UM-SCC-22A, UM-SCC-14A and UM-SCC-1) compared to non-cancerous keratinocytes using quantitative-polymerase chain reaction (Q-PCR). Consistent with HNSCC primary tumours, CerS1 was down-regulated about 10–20-fold in HNSCC cell lines compared to normal human epidermal primary keratinocytes (NHEK) or HPV-E6/E7-immortalized human keratinocytes controls (Fig 1A and B). CerS6 mRNA was similar in HNSCC cell lines and immortalized keratinocytes (Fig 1B). Down-regulation of CerS1 was also associated with lower C18-ceramide, measured with liquid chromatography/mass spectrometry (LC/MS/MS), in UM-SCC-1 and -22A (Fig 1C) compared to immortalized keratinocytes (about 90 and 50%, respectively). Thus, these studies indicate that CerS1/C18-ceramide, but not CerS6/C16-ceramide is down-regulated in HNSCC cancer cell lines compared to non-cancerous keratinocytes, consistent with decreased CerS1/C18-ceramide in HNSCC tumour compared to non-cancerous head and neck tissues (Karahatay et al, 2007; Koybasi et al, 2004).

Bottom Line: Moreover, an alternatively spliced variant CerS1 mRNA (CerS1-2) was detected mainly in cancer cells or primary tumour tissues compared to controls, which was targeted by miR-574-5p for degradation.Interference with HDAC1 and miR-574-5p reconstituted CerS1-2 expression and C(18) -ceramide generation in multiple human cancer cell lines, which subsequently inhibited proliferation and anchorage-independent growth.Accordingly, knockdown of CerS1 partially protected cancer cells from MS-275/miR-574-5p siRNA-mediated growth inhibition.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, SC, USA.

Show MeSH
Related in: MedlinePlus