Mid-region parathyroid hormone-related protein (PTHrP) binds chromatin of MDA-MB231 breast cancer cells and isolated oligonucleotides “in vitro”

Abstract

We have previously shown that PTHrP(38-94)-amide restrains growth and invasion "in vitro", causes striking toxicity and accelerates death of some breast cancer cell lines, the most responsive being MDA-MB231 whose tumorigenesis was also attenuated "in vivo". PTHrP(38-94)-amide contains the domain implicated in the nuclear import of PTHrP. Although the nucleus was identified as a destination for mid-region PTHrP, evidence for direct DNA-binding capability is lacking to date. Here, we examined the localization of PTHrP(38-94)-amide within MDA-MB231 cells and within metaphase spread preparations and characterized its DNA-binding properties, employing a combination of immunocytochemical, cytogenetic, "whole genome"/conventional PCR, EMSA and DNase footprinting techniques. The results obtained: (i) show that PTHrP(38-94)-amide gains access to the nuclear compartment of MDA-MB231 cell; (ii) demonstrate that PTHrP(38-94)-amide is a DNA-binding peptide; and, (iii) represent the first data to date on the potential molecular targets in both cellular chromatin and isolated oligonucleotides "in vitro".

Midregion PTHrP regulates Rip1 and caspase expression in MDA-MB231 breast cancer cells

“…More recently, evidence was produced that midregion PTHrP is imported in the nucleoplasm of cultured MDA-MB231 cells, and that ''in vitro'' it can bind chromatin of metaphase spread preparations and also an isolated 20-mer oligonucleotide, thereby appearing endowed with a putative transcription factor-like DNA-binding ability [23]. This is consistent with data indicating that midregion PTHrP possesses a nuclear targeting signal (NTS) encompassing aminoacids 87-107 and that the lysine-rich 84-93 domain is per se able to direct importin b/Ran GTPase-mediated import of to the nucleoplasm under the control of phosphorylation/dephosphorylation events in T 85 [24, for review].…”

PTHrP Produced by Myeloma Plasma Cells Regulates Their Survival and Pro-Osteoclast Activity For Bone Disease Progression

“…This approach is in line with the work by other authors to obtain a detectable effect of PTHrP and its fragments in in vitro studies. (19,41,57) In conclusion, we have identified novel pathogenetic functions of PTHrP in MM related to tumor survival, proliferation, and OC hyperactivation. Because serum elevation of PTHrP in patients with solid tumors produces severe skeleton devastation and leads to a poor prognosis, a systematic analysis of PTHrP levels in MM patients would be advisable, to determine whether this peptide is a novel marker of disease progression.…”

“…Indeed, the NLS þ mid-region (aa 38-94) is proteolytically degraded in cytoplasm and internalized by nuclei where this fragment primes in an intracrine fashion the proliferation of MM cells by its complementarity to the 5 0 -GAGTAGAATTCTAA-TATCTC-3 0 DNA sequence. (19) Other authors, however, have suggested that, to induce a similar effect, the NLS subunit may be transported and internalized in nuclei by cytoplasmic importin-b and that this mechanism is protective for tumor cells in preventing their apoptosis or anoikis induced by starvation. (41,42,46) By contrast, an opposite proapoptogenic effect has been ascribed to the NLS þ mid-region (aa 38-94) in cell kinetic analysis in breast cancer.…”

“…To visualize PTHrP nuclear translocation by proliferating MM cells, we completed additional experiments using RPMI 8226, both unstimulated and stimulated with 1 nM PTHrP mid-region including NLS (aa 38-94), in lowering serum concentration cultures, namely 10% to 0% of fetal calf serum (FCS), as described. (19) Subsequent analysis was completed by confocal microscopy (DM IRE2; Leica Microsystems, Wetzlar, Germany) after incubation with rabbit antiPTHrP mid-region (aa 34-53) (Calbiochem, Merck Chemicals Limited, Nottingham, UK; cod. PC09), secondary antibody and nuclei staining with 1 mM TO-PRO-3 Iodide (Invitrogen, Milan, Italy).…”

“…(2) The mid-region (aa 38-106) has been reported to contain the nuclear localization sequence (aa 87-94) (NLS) that binds DNA in nuclei with potential effects on cell proliferation, because NLS À/À mice show accelerated apoptosis in most tissues, which is associated with delayed growth, senescence, and early death. (19)(20)(21) Last, the COOH-terminal subunit (aa 107-139), also known as osteostatin, has been described as a negative regulator of signals induced by the NH 2 -terminal region and an inhibitor of the osteoclast activity in rats. (22) The functional role of PTHrP fragments in human pathophysiology has been intensively investigated and their generation by posttranslational proteolysis of members of the subtilisin family, has been definitely assessed.…”