Rosalia Sirchia

Midregion Parathyroid Hormone‐Related Protein Inhibits Growth and Invasion In Vitro and Tumorigenesis In Vivo of Human Breast Cancer Cells

Parathyroid hormone‐related protein (PTHrP) is critical for normal mammary development and is overexpressed by breast cancers. PTHrP is a peptide hormone that undergoes extensive post‐translational processing, and PTHrP(38–94)‐amide is one of the mature secretory forms of the peptide. In this study, we explored the effect of PTHrP(38–94)‐amide in a panel of six breast cancer cell lines “in vitro” and in MDA‐MB231 cells “in vivo” specifically examining cell viability, proliferation, invasiveness, and growth in nude mice. PTHrP(38–94)‐amide markedly inhibited proliferation and also caused striking toxicity and accelerated cell death in breast cancer cells. In addition, direct injection of PTHrP(38–94)‐amide into MDA‐MB231 breast cancer cells passaged in immunodeficient mice produced a marked reduction in tumor growth. These studies (i) indicate breast cancer cells are one of the few tissues in which specific effects of midregion PTHrP have been established to date, (ii) support a role for midregion secretory forms of PTHrP in modulating not only normal but also pathological mammary growth and differentiation, (iii) add further evidence for the existence of a specific midregion PTHrP receptor, and (iv) provide a novel molecule for modeling of small molecule analogues that may have anti‐breast cancer effects.

Type V collagen regulates the expression of apoptotic and stress response genes by breast cancer cells.

Type V collagen is a “minor” component of normal human breast stroma, which is subjected to over‐deposition in cases of ductal infiltrating carcinoma (DIC). We reported that, if used as a culture substrate for the DIC cell line 8701‐BC, it exhibited poorly‐adhesive properties and restrained the proliferative and motile behavior of the cell subpopulation able to attach onto it. Moreover, this collagen species was able to trigger DNA fragmentation and impair survival of 8701‐BC cells. In this study, we have extended our investigation with the aim to obtain further evidence that the death induced by type V collagen was of the apoptotic type by (i) microscopic detection and quantitation of Apoptag‐labeled cells, (ii) analysis of the expression levels of selected genes coding for apoptosis‐linked factors, caspases, and stress‐response proteins by conventional and semi‐quantitative multiplex PCR, and (iii) evaluation of the extent of caspase activation by chromogenic assay. We report here that type V collagen is able to determine an increase in the percentage of Apoptag‐positive cells, to up‐regulate Bcl‐xS, Bad, Dap kinase, hsf‐1, mthsp75, caspase‐1, ‐5, ‐8, ‐9, and ‐14, whilst down‐regulating Bcl‐2, Bcl‐xβ, and hsp60. Treatment of cell lysates with chromogenic tetrapeptide substrates specific for caspase‐1, ‐5, ‐8, and ‐9 demonstrated a marked increase of enzymatic activity in the presence of type V collagen. Our data validate 8701‐BC cell line as a suitable “in vitro” model for further and more detailed studies on the molecular mechanisms of the death response induced by type V collagen on primary DIC cells.

Cadmium as a transcriptional modulator in human cells

Cadmium (Cd) is an underground mineral widely used in the steel industry, in plastics, and as a component of batteries. It is an industrial and environmental pollutant released as an air contaminant from fertilizers and, more prominently, in the form of wastewater. Food, drinking water, and, mainly, inhalation of smoke from cigarettes are sources of daily exposure of humans to the heavy metal. Although Cd has no known useful function for humans as well as other organisms, it appears to evoke in cells a number of responses that involve not only death signaling but also protective reactions against the toxicity. This finding prompted a number of experimental studies aimed to elucidate the cellular and molecular aspects of Cd-dependent regulation of gene expression and signal transduction pathways in different model system. Here, the authors briefly review the role of Cd as a transcriptional regulator in diverse cytotypes of human origin, focusing in particular on its effects on two classes of genes, i.e., stress-response genes such as metallothioneins (MTs), heme oxygenase, and heat shock proteins (hsps), and apoptosis-related genes, but giving also an overview of many other examples of genes involved in cell metabolism and both intracellular and extracellular signalization whose expression levels are controlled by Cd.

PTHrP [67-86] regulates the expression of stress proteins in breast cancer cells inducing modifications in urokinase-plasminogen activator and MMP-1 expression

It was previously reported that a midregion domain of parathyroid hormone-related protein (PTHrP), that is, [67-86]-amide, is able to restrain growth and promote matrigel penetration by the 8701-BC cell line, derived from a biopsy fragment of a primary ductal infiltrating carcinoma of the human breast, and that cell invasion in vitro is drastically impaired by inactivation of urokinase-plasminogen activator (uPa). In this study we started a more detailed investigation of the possible effects on gene expression arising from the interaction between PTHrP [67-86]-amide and 8701-BC breast cancer cells by a combination of conventional-, differential display-and semi-quantitative multiplex-polymerase chain reaction (PCR) assays. We present here the first evidence that the upregulation of some stress-related genes, most noticeably heat shock factor binding protein-1 (hsbp1) and heat shock protein 90 (hsp-90), is involved in the acquisition of an in vitro more invasive phenotype by cells treated with midregion PTHrP. This is conceivably accomplished by sequestering and inactivating heat shock factor-1 (hsf1) which is able to recognize Ets transcription-factor-binding sites present in some gene promoters, such as those of uPa and matrix metalloprotease-1 (MMP-1). In fact, our data show that incubation of PTHrP [67-86]-amide-treated cells with either antisense hsbp1-oligonucleotide or geldanamycin, an hsp90-inactivating antibiotic, results in downregulation of uPa and upregulation of MMP-1, and in a prominent inhibition of cell invasion in matrigel-containing Transwell chambers. Alternatively, incubation of untreated 8701-BC cells with quercetin, a flavonoid known to decrease the amount of free hsf1, is found to induce upregulation of uPa and downregulation of MMP-1, and an increase of matrigel invasion by cells, thus providing further supporting data of the involvement of hsf unavailability on the modulation of uPa and MMP-1 expression and on cell invasive behaviour. These studies confirm a previous postulate that over-secretion of uPa, rather than of other extracellular proteases, is a primary condition for the increase of invasive activity triggered by PTHrP [67-86]-amide in vitro, and support a role for midregion forms of PTHrP in potentially affecting pathological mammary growth and differentiation. They also identify two new key protagonists in the complex scenario of breast tumor cell invasiveness in vitro, that is, hsbp1 and hsp90, which deserve further and more extensive studies as potential and attractive molecular targets for anti-breast cancer treatments.

Cadmium regulation of apoptotic and stress response genes in tumoral and immortalized epithelial cells of the human breast

Cadmium (Cd) is a widely-disseminated metal which can be imported and accumulated in living cells thereby drastically interfering with their biological mechanisms. Increasing interest has been recently focused on the elucidation of the cellular and molecular aspects of Cd-dependent regulation of gene expression and signal transduction pathways in different model system. Concerning breast cancer, very limited studies have been produced so far on the role played by Cd on estrogen receptor-negative human breast cancer cells, that are expected to be insensitive to the already-proven metallo-estrogenic effect exerted by Cd on the estrogen receptor-positive cell counterparts. Here, we have examined the effects of long-term (96 h) exposure of estrogen receptor-negative MDA-MB231 malignant adenocarcinoma cells to CdCl(2) at 5 microM concentration, corresponding to the IC(50) for this time of incubation, by evaluating the expression levels of genes coding for stress response factors (e.g. heat shock proteins and metallothioneins), and for apoptosis-related factors and enzymes. In parallel, we tested the gene expression pattern of immortalized HB2 breast epithelial cells, taken as non-tumoral counterpart, after the same exposure to the metal which instead did not exert any change in their cell number with respect to controls. Our cumulative results indicate that, whilst HB2 cells appear to activate defense mechanisms against metal stress principally via metallothionein massive up-regulation and appearance of the spliced form of XBP-1 message, MDA-MB231 cells seem to couple the onset of a protective reaction (e.g. up-regulation of hsp27 and metallothioneins) to the switching-on of new intracellular pathways directing cells to a kind of death which shares several aspects with the apoptotic program, such as down-regulation of Bcl-2 and over-expression of Dap kinase and several caspases.

T47-D cells and type V collagen: A model for the study of apoptotic gene expression by breast cancer cells

Abstract We have previously reported that type V collagen is a poorly adhesive, anti-proliferative and motility-inhibitory substrate for the 8701-BC breast cancer cell line, which also triggers DNA fragmentation and impairs survival of the same cell line. In the present work we have extended to other breast cancer cell lines (T47-D, MDA-MB231, Hs578T) our investigation of type V collagen influence on the DNA status and cell survival, also examining whether adhesion and growth of cells on this collagen substrate could exert some effect on the expression level of selected apoptosis related genes. We report here that, among the cell lines tested, only T47-D is responsive to the deathpromoting influence of type V collagen. In addition, the latter induces changes in gene expression by upregulating p53, Waf-1, Cas, Dap kinase and caspases 1, -5 and -14 and downregulating Bcl-2. Our data validate the T47-D line as a suitable in vitro model for further and more detailed studies on the molecular mechanisms of the death response induced by type V collagen on mammary tumor cells.

Short-term exposure to cadmium affects the expression of stress response and apoptosis-related genes in immortalized epithelial cells from the human breast

It is known that cadmium (Cd) evokes cell responses that not only involve protective reactions against toxicity but also induces cell death. Increasing interest has been recently focused on the elucidation of the cellular and molecular aspects of Cd-dependent regulation of gene expression in different model systems. Here, we examined the effects of short-term (24h) exposure of immortalized non-tumoral HB2 cells from human breast epithelium to CdCl(2) at 50 microM concentration, corresponding to the IC(50) for this time of incubation. The possible occurrence of apoptosis-related events was evaluated via analysis of the physical state of the DNA and of the membrane localization of phosphatydilserine. We also checked the pattern of expression of stress response genes, such as those coding for heat shock proteins and metallothioneins, and of genes coding for factors and enzymes involved in the onset of apoptosis. Our results indicate that although metal treatment appears to induce a non-apoptotic type of cell death, Cd can be also regarded as an active transcriptional modulator for this cell line.

Mid-region parathyroid hormone-related protein (PTHrP) and gene expression of MDA-MB231 breast cancer cells

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, for which tumorigenesis was also attenuated in vivo. We have also demonstrated that mid-region PTHrP gains access to the nuclear compartment of these cells and displays DNA-binding properties in vitro by recognizing targets in both cellular chromatin and isolated oligonucleotides. Here, we examined whether PTHrP(38–94) amide was able to modulate gene expression of MDA-MB231 cells, employing a combination of conventional, differential display and semi-quantitative multiplex PCR techniques. The results obtained provide first evidence that PTHrP(38–94) amide can affect gene expression in tumor cells, identifying A4-differentiation protein/PLP2 as up-regulated, and HOX7/MSX1, COX6C, FZD6, OXR1 and TMCO4 as down-regulated genes in treated cells, and suggest that the cytotoxic activity of the peptide can be ascribed, at least in part, to such transcriptional reprogramming.

Tumor cell-collagen interactions: Identification and semi-quantitative evaluation of selectively-expressed genes by combination of differential display- and multiplex-PCR

It is widely acknowledged that the presence of extracellular matrix components as substrates can drastically modulate the phenotype and gene expression of cultured cells, including tumor cells. A number of published reports indicated that substrates made from two peculiar collagen species, i.e. type V and OF/LB, which are abnormally deposited in the stroma of primary ductal infiltrating carcinoma (d.i.c.) of the breast “in vivo,” were able to exert marked and opposite effects on “in vitro” viability, growth and invasiveness of the 8701-BC cell line, isolated from d.i.c.-affected breast epithelium. To complement such functional data on the effect of cell-collagen interactions with information at molecular level, we have utilized a combination of differential display- and semi-quantitative multiplex-PCR techniques with the aim of detecting variations in the expression levels of selected genes by cells maintained in either culture condition. Here we report some prototypical data on the identification and semi-quantitation of three of the differentially-amplified PCR products found, i.e.HSP2A andMSF-B which are up-regulated in cells grown onto OF/LB collagen substrate, andSRCAP which is prominently down-regulated in the presence of type V collagen substrate. This protocol represents a powerful tool for evaluating changes in the levels and patterns of gene expression which can be theoretically adapted to any experimental model system.

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

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”.