A class of DNA-binding peptides from wheat bud causes growth inhibition, G2 cell cycle arrest and apoptosis induction in HeLa cells

Abstract

Background: Deproteinized DNA from eukaryotic and prokaryotic cells still contains a low-molecular weight peptidic fraction which can be dissociated by alkalinization of the medium. This fraction inhibits RNA transcription and tumor cell growth. Removal from DNA of normal cells causes amplification of DNA template activity. This effect is lower or absent in several cancer cell lines. Likewise, the amount of active peptides in cancer cell DNA extracts is lower than in DNA preparation of the corresponding normal cells. Such evidence, and their ubiquitous presence, suggests that they are a regulatory, conserved factor involved in the control of normal cell growth and gene expression.

Results: We report that peptides extracted from wheat bud chromatin induce growth inhibition, G2 arrest and caspase-dependent apoptosis in HeLa cells. The growth rate is decreased in cells treated during the S phase only and it is accompanied by DNA damage and DNA synthesis inhibition. In G2 cells, this treatment induces inactivation of the CDK1-cyclin B1 complex and an increase of active chk1 kinase expression.

Conclusion: The data indicate that the chromatin peptidic pool inhibits HeLa cell growth by causing defective DNA replication which, in turn, arrests cell cycle progression to mitosis via G2 checkpoint pathway activation.

Figure 1

Cell growth following incubation with the peptide fraction. The cells were incubated for 24 hours (white circles) and 48 hours (black circles) with different concentration of the peptidic pool (A) and for different times with 5 μg/ml of the peptide fraction (B). The results are expressed as percentage of cell number in treated cultures.

Figure 2

Chromatin peptides induce apoptosis in HeLa cells. A: apoptosis level measured by DNA fragmentation ELISA after 12, 24 and 48 hours of treatment with 5 μg/ml of peptide pool. (black bars) control cells; (grey bars) treated cells. B: analysis of proteins involved in apoptosis induced by chromatin peptides. Whole cell extracts were prepared from treated (24 h) and untreated HeLa cells. The same amount of proteins was assayed by PAGE and western blotting using antibodies specific for: (a) cleaved caspase-7, (b) cleaved caspase-9, (c) Bax, (d) Bcl-2 and (f) cleaved PARP. C: densitometric analysis of the immunoblots. The values are expressed as percentage of the control.

Figure 3

G₂ arrest of HeLa cells following the treatment with the peptidic pool. A: DNA distributions for control (left) and treated (right) HeLa cells after treatment with 5 μg/ml of the peptide pool for 12 h. B: Cell cycle changes after 12, 24 and 48 hours of incubation with 5 μg/ml of peptide pool. Percentage of cells in: (black) G₁ phase; (white) S phase; (grey) G₂/M phase; c: control cells; t: treated cells. C: Distribution of the cells in the mitosis phases following incubation for 24 hours with (grey bars) 2.5 μg/ml or with (white bars) 5 μg/ml of peptides; (black bars) control cells.

Figure 4

Effects of the chromatin peptides on the various phases of the cell cycle. A: Cell cycle analysis by FACS of unsynchronized cells and of synchronized cells at 1, 6 and 8 hours after removal of the thymidine block. (black) percentage of cells in G₁ phase; (white) percentage of cells in S phase; (grey) percentage of cells in G₂/M phase. B: Cell growth after treatment with different concentration of peptide fraction in the various phases of cell cycle as described in the text; (black bars) treatment during S phase; (grey bars) treatment during S+G₂ phases; (white bars) treatment during G₂ phase as described in the text.

Figure 5

Image of DNA migration obtained by fluorescence microscopy following single cell electrophoresis performed on S phase cells. A: control cells; B: cells incubated for 2 hours with 5 μg/ml of peptide fraction.

Figure 6

Induction of the G₂ checkpoint control by chromatin peptides. A: Expression of cyclin B1 at 12 and 24 hours of peptide fraction treatment. (black bars) control cells; (grey bars) treated cells. The analyses were performed by flow cytometry as described in Materials and Methods. B: Cdc2-cyclin B1 activity determined by ELISA in nuclear extracts obtained from G₂ cells. C: Western Blot analysis performed in nuclear extracts obtained from G₂ cells. a: inactive phospho-Tyr15 cdc2-cyclin B1 complex; b: active phospho-Ser345 chk1. D: densitometric analysis of the immunoblotting. The values are expressed as percentage of the control. c: control cells; t: treated cells.