Expression of a novel peptide derived from PCNA damages DNA and reverses cisplatin resistance

Abstract

Background: An 8 amino acid peptide sequence derived from proliferating cell nuclear antigen (PCNA) has been shown to effectively kill several breast cancer and neuroblastoma cell lines when added exogenously to cell cultures.

Methods: In this study, the expression of the 8 amino acid peptide sequence (caPeptide) was placed under control of a tetracycline responsive promoter in MDA-MB-231 cells.

Results: Endogenous expression of the peptide resulted in an increase in genomic DNA damage. CaPeptide induction combined with treatment of sublethal doses of cisplatin resulted in a marked increase in death of the cisplatin-resistant MDA-MB-231 cell line. CaPeptide was found to interact with POLD3, one of the subunits of DNA polymerase delta necessary for binding to PCNA.

Conclusion: These results suggest an important line of inquiry into the possible role that caPeptide might play in the reversal of cisplatin resistance in breast and other cancers. This is of particular interest in those cancers where cisplatin is the first line of chemotherapy and where the acquisition of resistance is a common malady.

Fig. 1

Loss of viability relative to scrambled control peptide in a panel of breast cancer cell lines treated with 60 μM R9-caPeptide for 72 h. Cell surface receptor status for each cell line is indicated below chart (N no, Y yes, “−” negative, “+” positive, “++” amplified)

Fig. 2

a Nucleotide sequence of caPeptide placed into expression vector along with the corresponding amino acid translation. The amino acids corresponding to a.a. 126–133 of caPCNA are underlined. The peptide sequence was followed by a three nuclear localization signal repeat (3 × NLS). b Diagram representation of expression vector used for creation of stable inducible cell population. Expression vector features include the tetracycline inducible promoter, pTight, caPeptide-3 × NLS followed by the co-translational cleavage moderator, 2a peptide and the fluorescent reporter gene, mcherry. Also present is a puromycin resistance selectable marker. c Bright field and fluorescence composite microscopy image showing Dox induction of caPeptide and reporter protein mcherry in 231-caPeptide cell lines. Induction results in a single transcript containing the coding information for the respective peptide and mcherry reporter which is then translated in a single ribosomal event but cleaved into peptide and reporter gene through the activity of the 2a peptide sequence. This results in equimolar expression of peptide and reporter gene. d Reverse transcription PCR of mRNA from Dox-treated and untreated 231-caPeptide cell populations. Reaction #1 used primers that annealed to the 5′ and 3′ ends of the mcherry sequence in the cDNA template. Reaction #2 used primers that annealed to the 5′ end of caPeptide and same 3′ end primer as reaction #1. The expected products are 770 and 930 bp, respectively. e Lysates of 231-v.c cells (vector control) and 231-flg-caPeptide cells metabolically labeled with tritiated lysine in the presence and absence of doxycycline were immunoprecipitated with an antibody specific to the flag tag (Anti-Flag) and an antibody developed by using the caPeptide sequence as an immunogen (Anti-caPCNA). The samples were separated by gel electrophoresis and visualized by autoradiography

Fig. 3

a 231-caPeptide cells labeled with tritiated lysine and treated with or without Dox were fractionated to separate cytoplasm from nucleus. To identify the fractions containing caPeptide, lysates were immunoprecipitated with anti-caPCNA antibody, separated by gel electrophoresis and imaged by autoradiography. To determine the success of fractionation, lysates were subjected to immunoblotting, and antibodies to Histone H3 and GAPDH were used to verify nuclear and cytoplasmic fractions. b Tritiated 231-caPeptide lysates treated with or without Dox were immunoprecipitated with antibody specific to POLD3. After gel electrophoresis, tritiated caPeptide was detected by autoradiography and equal amounts of immunoprecipitated POLD3 in “−”and “+” Dox samples was verified by immunoblotting

Fig. 4

Comet assay of 231-caPep cells that were left untreated, treated for 24 h with either 5 μM CDDP (CDDP) or 1 μg/ml Dox (CaPep), or treated 24 h with both 5 μM CDDP and 1 μg/ml Dox (CDDP + CaPep). Cells were scored as having no damage, low damage, medium damage or high damage. a Representative examples of cells in three categories of damage. b Compiled results of the different treatments and categories of damage (FC fold change, p p value of Student’s t test)

Fig. 5

a Growth curve on 231-caPep cells treated with CDDP, with and without Dox-induced caPeptide expression. 231-caPep cells were plated at 1 × 10⁵ cells per 60-mm plate on Day 0 for each condition and time point. Each sample was plated in triplicate. On Day 1, Dox at 1 μg/ml and CDDP at 2, 3.5 or 5 μM was added to appropriate samples. Dox was replenished every other day and CDDP and media changed every fourth day. Samples were collected and counted on days 1, 5, 9 and 13. Cells treated with CDDP and expressing caPeptide began dying at a dose-dependent rate after replenishing media, Dox and CDDP on Day 5. b Clonogenic assay on 231-caPep cells with or without induced caPeptide expression treated with increasing doses of CDDP. 231-caPep was treated with or without 1 μg/ml of Dox and 0, 10, 20, 30, 45 and 60 μM of CDDP for 24 h. One thousand cells of each condition were plated in triplicate. Dox continued to be added to “+Dox” samples at a rate of 1 μg/ml Dox every other day. After 5 days, plates were stained and colonies counted. CaPeptide expressing cells were more susceptible to CDDP with the IC50 of caPeptide expressing cells dropping by 50 %. c Cell cycle analysis of 231-caPep cells expressing caPeptide and treated with a low level of CDDP. 231-caPep and 231-v.c. cells were treated overnight with 10 μM CDDP and 1 μg/ml Dox. Flow cytometry was used to analyze the DNA content of prepared samples. Cells expressing caPeptide had a fivefold increase in apoptotic cells

Fig. 6

MDA-MB-231 cells were treated with R9-cc-caPeptide for 0, 12, 24, 48 and 72 h. The cells were collected, fixed, permeabilized and stained with fluorescent antibodies to γH2AX and the 89-kDa-cleaved fragment of PARP. Flow cytometry was used to analyze the samples