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Cisplatin fails to induce puma mediated apoptosis in mucosal melanomas.

Fritsche MK, Metzler V, Becker K, Plettenberg C, Heiser C, Hofauer B, Knopf A - Oncotarget (2015)

Bottom Line: IHC and WB confirmed high p53 expression in all melanomas.Hipk2 and Gadd45 showed significantly higher expressions in CM (p < 0.005; p = 0.004).WB failed to detect Puma in MM, while Cdk1 regulation occurred exclusively in MM.

View Article: PubMed Central - PubMed

Affiliation: Technische Universität München, Hals-Nasen-Ohrenklinik und Poliklinik, 81675 München, Germany.

ABSTRACT

Objectives: Mucosal melanomas (MM) are aggressive subtypes of common melanomas. It remains unclear whether limitations in their resectability or their distinctive molecular mechanisms are responsible for the aggressive phenotype.

Methods: In total, 112 patients with cutaneous melanomas (CM) and 27 patients with MM were included. Clinical parameters were analysed using Chi square, Fisher exact and student's t-test. Survival rates were calculated by Kaplan-Meier. Analysis of p53, p21, Mdm2, Hipk2, Gadd45, Puma, Bax, Casp9 and Cdk1 via quantitative PCR and immunohistochemistry (IHC) was performed. TP53 induction after cisplatin treatment was analysed in 10 cell lines (melanocytes, four MM and five CM) using western blot (WB) and qPCR.

Results: The overall/recurrence-free survival differed significantly between MM (40 months and 30 months) and CM (90 months and 107 months; p < 0.001). IHC and WB confirmed high p53 expression in all melanomas. Hipk2 and Gadd45 showed significantly higher expressions in CM (p < 0.005; p = 0.004). QPCR and WB of wild-type cell lines demonstrated no differences for p53, p21, Mdm2, Bax and Casp9. WB failed to detect Puma in MM, while Cdk1 regulation occurred exclusively in MM.

Conclusions: The aggressive phenotype of MM did not appear to be due to differential expressions of p53, p21, Mdm2, Bax or Casp9. A non-functional apoptosis in MM may have further clinical implications.

No MeSH data available.


Related in: MedlinePlus

Quantitative PCR of the p53 pathway in primary cells and cell lines without treatment and after 24 h incubation with 8 μM cisplatinResults were normalized to GAPDH and shown as fold induction compared to CM. MM: Mucosal melanoma; CM: Cutaneous melanoma. Cis: Cisplatin.
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Figure 3: Quantitative PCR of the p53 pathway in primary cells and cell lines without treatment and after 24 h incubation with 8 μM cisplatinResults were normalized to GAPDH and shown as fold induction compared to CM. MM: Mucosal melanoma; CM: Cutaneous melanoma. Cis: Cisplatin.

Mentions: QPCR of untreated MM cells and CM cell lines revealed different expression patterns of analysed genes due to the underlying p53 mutation status. Primary MM cells and melanoma cell lines harbouring wild-type p53 demonstrated a significantly lower TP53 mRNA expression when compared with primary melanocytes. The lowest TP53 mRNA levels were determined for p53-knockout cell line IGR-37. Parallel with p53 mRNA levels, significantly decreased mRNA levels were detected for p53 stabilizing HIPK2 in all p53 wild-type cell lines and IGR-37. No differences were observed for melanoma cell lines expressing mutated p53. Direct p53 target gene CDKN1A demonstrated significantly lower expressions for all tumour cell lines when compared with primary melanocytes. A significant and stepwise decrease can be observed in MM cells, from melanoma cell lines harbouring wild-type p53 to melanoma cell lines with mutated p53. GADD45A mRNA was expressed equally in primary melanocytes and MM cells; a significantly higher expression than that of CM cell lines. BBC3 and BAX had significantly lower expression in MM and CM cells. The lowest levels demonstrated p53- knockout IGR-37. Analysis of CASP9 demonstrated a significantly decreased mRNA expression when compared with primary melanocytes. No differences were observed for CDK1 expression levels in MM and CM, except IGR-37 (Figure 3). To investigate the functional integrity of the p53 downstream, cells were treated with 8 μM cisplatin. There was no regulation of TP53 in the analysed cell lines. All cell lines harbouring wild-type p53 demonstrated an upregulation of MDM2, with the highest expression in melanocytes and MM. HIPK2 was downregulated in melanocytes and CM cell lines that expressed mutated p53. Cell cycle regulation CDKN1A was upregulated in all cell lines after treatment with cisplatin, while GADD45A was upregulated only in melanocytes. BBC3 mRNA expression increased in CM cell lines with wild-type p53 and in MM. QPCR failed to demonstrate a regulation BAX or CASP9. A CDK1 downregulation was exclusively seen in MM (Figure 3).


Cisplatin fails to induce puma mediated apoptosis in mucosal melanomas.

Fritsche MK, Metzler V, Becker K, Plettenberg C, Heiser C, Hofauer B, Knopf A - Oncotarget (2015)

Quantitative PCR of the p53 pathway in primary cells and cell lines without treatment and after 24 h incubation with 8 μM cisplatinResults were normalized to GAPDH and shown as fold induction compared to CM. MM: Mucosal melanoma; CM: Cutaneous melanoma. Cis: Cisplatin.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496404&req=5

Figure 3: Quantitative PCR of the p53 pathway in primary cells and cell lines without treatment and after 24 h incubation with 8 μM cisplatinResults were normalized to GAPDH and shown as fold induction compared to CM. MM: Mucosal melanoma; CM: Cutaneous melanoma. Cis: Cisplatin.
Mentions: QPCR of untreated MM cells and CM cell lines revealed different expression patterns of analysed genes due to the underlying p53 mutation status. Primary MM cells and melanoma cell lines harbouring wild-type p53 demonstrated a significantly lower TP53 mRNA expression when compared with primary melanocytes. The lowest TP53 mRNA levels were determined for p53-knockout cell line IGR-37. Parallel with p53 mRNA levels, significantly decreased mRNA levels were detected for p53 stabilizing HIPK2 in all p53 wild-type cell lines and IGR-37. No differences were observed for melanoma cell lines expressing mutated p53. Direct p53 target gene CDKN1A demonstrated significantly lower expressions for all tumour cell lines when compared with primary melanocytes. A significant and stepwise decrease can be observed in MM cells, from melanoma cell lines harbouring wild-type p53 to melanoma cell lines with mutated p53. GADD45A mRNA was expressed equally in primary melanocytes and MM cells; a significantly higher expression than that of CM cell lines. BBC3 and BAX had significantly lower expression in MM and CM cells. The lowest levels demonstrated p53- knockout IGR-37. Analysis of CASP9 demonstrated a significantly decreased mRNA expression when compared with primary melanocytes. No differences were observed for CDK1 expression levels in MM and CM, except IGR-37 (Figure 3). To investigate the functional integrity of the p53 downstream, cells were treated with 8 μM cisplatin. There was no regulation of TP53 in the analysed cell lines. All cell lines harbouring wild-type p53 demonstrated an upregulation of MDM2, with the highest expression in melanocytes and MM. HIPK2 was downregulated in melanocytes and CM cell lines that expressed mutated p53. Cell cycle regulation CDKN1A was upregulated in all cell lines after treatment with cisplatin, while GADD45A was upregulated only in melanocytes. BBC3 mRNA expression increased in CM cell lines with wild-type p53 and in MM. QPCR failed to demonstrate a regulation BAX or CASP9. A CDK1 downregulation was exclusively seen in MM (Figure 3).

Bottom Line: IHC and WB confirmed high p53 expression in all melanomas.Hipk2 and Gadd45 showed significantly higher expressions in CM (p < 0.005; p = 0.004).WB failed to detect Puma in MM, while Cdk1 regulation occurred exclusively in MM.

View Article: PubMed Central - PubMed

Affiliation: Technische Universität München, Hals-Nasen-Ohrenklinik und Poliklinik, 81675 München, Germany.

ABSTRACT

Objectives: Mucosal melanomas (MM) are aggressive subtypes of common melanomas. It remains unclear whether limitations in their resectability or their distinctive molecular mechanisms are responsible for the aggressive phenotype.

Methods: In total, 112 patients with cutaneous melanomas (CM) and 27 patients with MM were included. Clinical parameters were analysed using Chi square, Fisher exact and student's t-test. Survival rates were calculated by Kaplan-Meier. Analysis of p53, p21, Mdm2, Hipk2, Gadd45, Puma, Bax, Casp9 and Cdk1 via quantitative PCR and immunohistochemistry (IHC) was performed. TP53 induction after cisplatin treatment was analysed in 10 cell lines (melanocytes, four MM and five CM) using western blot (WB) and qPCR.

Results: The overall/recurrence-free survival differed significantly between MM (40 months and 30 months) and CM (90 months and 107 months; p < 0.001). IHC and WB confirmed high p53 expression in all melanomas. Hipk2 and Gadd45 showed significantly higher expressions in CM (p < 0.005; p = 0.004). QPCR and WB of wild-type cell lines demonstrated no differences for p53, p21, Mdm2, Bax and Casp9. WB failed to detect Puma in MM, while Cdk1 regulation occurred exclusively in MM.

Conclusions: The aggressive phenotype of MM did not appear to be due to differential expressions of p53, p21, Mdm2, Bax or Casp9. A non-functional apoptosis in MM may have further clinical implications.

No MeSH data available.


Related in: MedlinePlus