Stimulation Of Cellular Proliferation Research Articles

Abstract P4-01-12: Short-term trastuzumab treatment increases oncogenic fitness in HER2 overexpressing breast cancer models

Abstract Overexpression of Human EGF Receptor 2 (HER2) is found in up to 20% of primary invasive breast cancers and is a marker of aggressive metastatic disease and poor prognosis. HER2 is a member of the transmembrane tyrosine kinase receptors family which includes EGFR (HER1), HER3 and HER4. Phosphorylation of tyrosine residues in the cytoplasmic domain upon homo- or heterodimerization results in stimulation of cellular proliferation, migration, angiogenesis, and inhibition of apoptosis. HER2 is deemed a preferred heterodimer partner and presents an excellent target for personalized medicine. Anti-HER2 monoclonal antibody trastuzumab (TZM) has been used in the clinic over the last decades and it is considered as one of the most successful targeted anti-cancer therapies. However, a large fraction of eligible patients displays either primary or acquired resistance to TZM treatment. In this study, we report that even relatively short 24h TZM exposure (TZM-priming) of AU565 HER2-overexpressing breast cancer cells results in rapid and profound alterations in HER receptors’ expression level and signaling, as well as upregulation of markers associated with drug resistance. We provide several lines of evidence to support this claim: Firstly, Western blot analysis of both monolayer AU565 cells incubated in the presence or absence of 20 μg/mL TZM (TZM priming) or human IgG (control), as well as of liquid overlay AU565 spheroids generated from TZM-primed or control cells, consistently shows significant increase of HER3 and pHER2 levels in the TZM-primed samples. Secondly, immunofluorescent analysis strongly indicates that TZM priming substantially upregulates HER3 and pHER2 levels both in 2D and 3D (spheroids) models. Importantly, this process is accompanied by rearrangement of endocytic markers Rab4, CD63, Sorl1 as well as Extra domain B fibronectin, found at the cells’ surface together with HER2 and HER3 upon TZM priming. A similar trend was also found in HER2-overexpressing ovarian cancer cells SKOV-3 subjected to short-term TZM treatment. Furthermore, spheroids made with TZM-primed AU565 cells display significantly faster cell proliferation compared to IgG-primed or untreated control counterparts, as measured via optical coherent tomography imaging and Imaris 3D rendering software. Finally, tumor xenografts implanted in athymic nude mice using TZM-primed AU565 cells display continuous growth in contrast to those generated from untreated cells. These results suggest that short-term TZM treatment may inadvertently increase oncogenic fitness via adaptation to TZM-HER2 cellular binding and subsequent disruption of dimerization and signaling. These results are consistent with reports indicating that regional HER2 expression heterogeneity appears to increase with TZM treatment. Moreover, these observations are especially concerning because radiolabeled TZM probes have been employed in PET imaging in HER2+ cancer patients. In summary, our results suggest that short-term TZM treatment may result in priming and selection for more aggressive cancer phenotype. Citation Format: Alena Rudkouskaya, Cassandra L Roberge, Lauren Elder, Kailie Matteson, David T Corr, Margarida Barroso. Short-term trastuzumab treatment increases oncogenic fitness in HER2 overexpressing breast cancer models [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-01-12.

Oxidative stress in bladder cancer: an ally or an enemy?

Bladder cancer (BC) is the most common cancer of the urinary tract and despite all innovations, remains a major challenge due to high morbidity and mortality. Genomic and epigenetic analyses allowed the discovery of new genes and pathways involved in the pathogenesis and regulation of BC. However, the effect on mortality has been modest and the development of new targets for BC treatment are needed. Recent evidence suggests that cancer cells are under increased stress associated with oncogenic transformation, with changes in metabolic activity and increased generation of reactive oxygen species (ROS). The increased amounts of ROS in cancer cells are associated with stimulation of cellular proliferation, promotion of mutations and genetic instability, as well as alterations in cellular sensitivity to anticancer agents. Since these mechanisms occur in cancer cells, there is a close link between oxidative stress (OS) and BC with implications in prevention, carcinogenesis, prognosis, and treatment. We address the role of OS as an enemy towards BC development, as well as an ally to fight against BC. This review promises to expand our treatment options for BC with OS-based therapies and launches this approach as an opportunity to improve our ability to select patients most likely to respond to personalized therapy.

Characterization of Cytokines and Proliferation Marker Ki67 in Cleft Affected Lip Tissue.

Backgroundandobjectives: Cleft lip palate takes the second place among all anomalies. The complex appearance of cytokines and proliferation markers has still not been clarified despite their possible crucial role in cleft tissue. Therefore, the aim of work was the detection of appearance of pro- and anti-inflammatory cytokines and proliferation marker Ki67, and their inter-correlations in cleft affected lip (CAL). Materials and Methods: The lip material was obtained from 16 children aged before primary dentition during plastic surgery. Control was obtained from 7 non-CAL oral tissue. Tissues were stained for IL-1, IL-4, IL-6, IL-8, IL-10 and Ki67 immunohistochemically. Non-parametric statistic, Mann–Whitney and Spearman’s coefficient were used. Results: All cytokines positive cells were observed more into the epithelium. Statistically significant difference was seen between epithelial IL-1, IL-10, IL-8 and Ki67 positive cells and IL-10-, IL-4-containing connective tissue cells in comparison to the control. Strong positive correlation was detected in CAL epithelium between IL-10 and IL-8, IL-10 and IL-4, IL-10 and IL-1, IL-1 and IL-8, IL-1 and IL-4, IL-4 and IL-8, IL-8 and Ki67, IL-10 and Ki67, but moderate—in connective tissue between IL-1 and IL-10, IL-1 and IL-4. Conclusions: The CAL epithelium is the main source for the interleukins. Rich similar expression of IL-1 and IL-10 suggests the balance between pro-and anti-inflammatory tissue response on basis of dysregulated tissue homeostasis (increase of IL-8). The correlations between the different ILs-1, -4, -8, -10 in CAL epithelium seem to indicate the self-protection compensatory mechanism for intensification of local inflammatory-immune response without involvement of IL-6. The correlations between Ki67 and cytokines indicate the involvement of IL-8 and IL-10 in stimulation of cellular proliferation. IL-4 and IL-10 expression from CAL connective tissue simultaneously to IL-1, IL-4 and IL-10 inter-correlations there suggests the intensification of local immune response regulated probably by main pro-inflammatory cytokine—IL-1.

Retrospective Proteomic Screening of 100 Breast Cancer Tissues.

The present investigation has been conducted on one hundred tissue fragments of breast cancer, collected and immediately cryopreserved following the surgical resection. The specimens were selected from patients with invasive ductal carcinoma of the breast, the most frequent and potentially aggressive type of mammary cancer, with the objective to increase the knowledge of breast cancer molecular markers potentially useful for clinical applications. The proteomic screening; by 2D-IPG and mass spectrometry; allowed us to identify two main classes of protein clusters: proteins expressed ubiquitously at high levels in all patients; and proteins expressed sporadically among the same patients. Within the group of ubiquitous proteins, glycolytic enzymes and proteins with anti-apoptotic activity were predominant. Among the sporadic ones, proteins involved in cell motility, molecular chaperones and proteins involved in the detoxification appeared prevalent. The data of the present study indicates that the primary tumor growth is reasonably supported by concurrent events: the inhibition of apoptosis and stimulation of cellular proliferation, and the increased expression of glycolytic enzymes with multiple functions. The second phase of the evolution of the tumor can be prematurely scheduled by the occasional presence of proteins involved in cell motility and in the defenses of the oxidative stress. We suggest that this approach on large-scale 2D-IPG proteomics of breast cancer is currently a valid tool that offers the opportunity to evaluate on the same assay the presence and recurrence of individual proteins, their isoforms and short forms, to be proposed as prognostic indicators and susceptibility to metastasis in patients operated on for invasive ductal carcinoma of the breast.

Peptide Agonists of Vasopressin V2 Receptor Reduce Expression of Neuroendocrine Markers and Tumor Growth in Human Lung and Prostate Tumor Cells.

Neuroendocrine tumors (NETs) comprise a heterogeneous group of malignancies that express neuropeptides as synaptophysin, chromogranin A (CgA), and specific neuronal enolase (NSE), among others. Vasopressin (AVP) is a neuropeptide with an endocrine, paracrine, and autocrine effect in normal and pathological tissues. AVP receptors are present in human lung, breast, pancreatic, colorectal, and gastrointestinal tumors. While AVP V1 receptors are associated with stimulation of cellular proliferation, AVP V2 receptor (V2r) is related to antiproliferative effects. Desmopressin (dDAVP) is a synthetic analog of AVP that acts as a selective agonist for the V2r, which shows antitumor properties in breast and colorectal cancer models. Recently, we developed a derivative of dDAVP named [V4Q5]dDAVP, which presents higher antitumor effects in a breast cancer model compared to the parental compound. The goal of present work was to explore the antitumor properties of the V2r agonist dDAVP and its novel analog [V4Q5]dDAVP on aggressive human lung (NCI-H82) and prostate cancer (PC-3) cell lines with neuroendocrine (NE) characteristics. We study the presence of specific NE markers (CgA and NSE) and V2r expression in NCI-H82 and PC-3. Both cell lines express high levels of NE markers NSE and CgA but then incubation with dDAVP diminished expression levels of both markers. DDAVP and [V4Q5]dDAVP significantly reduced proliferation, doubling time, and migration in both tumor cell cultures. [V4Q5]dDAVP analog showed a higher cytostatic effect than dDAVP, on cellular proliferation in the NCI-H82 cell line. Silencing of V2r using small interfering RNA significantly attenuated the inhibitory effects of [V4Q5]dDAVP on NCI-H82 cell proliferation. We, preliminarily, explored the in vivo effect of dDAVP and [V4Q5]dDAVP on NCI-H82 small cell lung cancer xenografts. Treated tumors (0.3 μg kg−1, thrice a week) grew slower in comparison to vehicle-treated animals. In this work, we demonstrated that the specific agonists of V2r, dDAVP, and [V4Q5]dDAVP displays antitumor capacity on different human models of lung and prostate cancers with NE features, showing their potential therapeutic benefits in the treatment of these aggressive tumors.

Abstract IA07: Targeting the cyclin D-CDK4/6 pathway for cancer therapy

Abstract The cyclin D-CDK4/6 kinase complex plays a pivotal role in the stimulation of cellular proliferation through phosphorylation and functional inactivation of the retinoblastoma protein (pRb), which leads to commitment of G1-phase cells to enter S phase, and ultimately to complete the mitotic cell cycle. Cancer cells frequently escape normal constraints on cell proliferation via persistent inactivation of pRb, through dysregulation of cyclin D-CDK4/6 kinase activity or loss of pRb expression. Interestingly, estrogen-positive (ER+) breast cancer cells rarely show loss of pRb, and hence rely on inappropriate activation of cyclin D-CDK4/6 to drive malignant proliferation. ER+ breast cancer therefore represents an enriched patient population in which to test the efficacy of cyclin D-CDK4/6 inhibition as a therapeutic strategy. Palbociclib, a selective cyclin D-CDK4/6 inhibitor, was approved by the FDA in 2015 for the treatment of women with advanced ER+/HER2- breast cancer. The drug is administered in combination with an anti-hormonal agent, which underscores the critical, collaborative roles of dysregulated estrogen receptor and cyclin D-CDK4/6 activities in driving tumor growth in this breast cancer subtype. This presentation will review the cellular pharmacology of palbociclib, with a particular emphasis on the phenotypic outcomes of combined estrogen receptor and cyclin D-CDK4/6 inhibition in human breast cancer cell lines. In addition, we will provide an update on the clinical activities of palbociclib in breast cancer, as well as prospects for therapeutic targeting of cyclin D-CDK4/6 activity in other cancer subtypes. Citation Format: Robert T. Abraham. Targeting the cyclin D-CDK4/6 pathway for cancer therapy. [abstract]. In: Proceedings of the AACR Precision Medicine Series: Cancer Cell Cycle - Tumor Progression and Therapeutic Response; Feb 28-Mar 2, 2016; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(11_Suppl):Abstract nr IA07.

Stimulation of Cellular Proliferation and Migration: Is It a Viable Measure of Photobiomodulation?

Stimulation of Cellular Proliferation and Migration: Is It a Viable Measure of Photobiomodulation?

Mechanisms of cellular proliferation stimulation under the effect of L-amino acids in the tissue culture of young and old rats

The statistical method of rank correlation was used to analyze the correlation of individual physicochemical characteristics of L-amino acids, as well as of dipole moments of the first pair of nucleotides of their codons, to the biological activity of amino acids, i.e. the stimulation of cellular proliferation. The experimental material was obtained from the organotypic tissues of ecto- and mesodermal origin in young and old rats. It was demonstrated that each of the stimulating amino acids acts 1.5–6.0 times less often in explants from old rats as compared to the explants from young rats. A weak correlation was detected between the stimulation of cellular proliferation and physicochemical characteristics (hydrophobicity, dipole moment and isoelectric point of the amino acids) in many tissues, but high and moderate correlation was detected in the brain subcortical explants, cerebellum and skin. However, a moderately close correlation was detected between the dipole moment of the first pair of nucleotides of their codons and the stimulating activity for explants of the brain subcortical structures (young and old rats), cerebellum and skin (young rats), and Spearman’s correlation coefficient (r s = 0.52; 0.45; 0.60; and 0.71, respectively). A moderate correlation was also detected for explants of the testis and cartilage. It can be suggested that some physicochemical and biochemical characteristics of amino acids correlate to their biological activity, which is especially remarkable in tissues derived from ectoderm.

Age-related molecular aspects of immunomodulating activity of peptides in the spleen

In this work, it was shown that short peptides, such as vilon, thymogen, crystagen and R-1, possess different age-associated immunoprotecting properties in the spleen. Both the R-1 and vilon peptides were found to activate T-helper cells. T-Helper activation by vilon was based on the inhibition of cellular apoptosis, whereas R-1 acted through positive stimulation of cellular proliferation and differentiation. Thymogen was found to be an activator of B-lymphocytes and acted through the inhibition of apoptosis, as well as by the stimulation of spleen cells proliferation. Crystagen was also found to activate the B-cell immunity system but had no effect on the processes of cellular renewal in the spleen during aging.

Metallothionein immunoexpression in selected benign epithelial odontogenic tumors

Odontogenic tumors exhibited variable biologica behaviors. Metallothionein (MT) is correlated with the cellular homeostasis of essential metals, cellular differentiation, and proliferation. The core goals of this study are (i) to report and to compare MT expression among benign epithelial odontogenic tumors; (ii) to correlate MT with cellular proliferation index; and (iii) to evaluate the influence of the inflammatory infiltrate on MT expression. Ten cases of solid ameloblastomas (SABs), 4 squamous odontogenic tumors (SOTs), 5 adenomatoid odontogenic tumors (AOTs), and 3 calcifying epithelial odontogenic tumors (CEOTs) were subjected to immunohistochemical to anti-MT, anti-Ki-67, and anti-PCNA. Statistical analysis was performed using BioEstat(®) 4.0. Metallothionein staining was found to be the highest in the SABs (93.1%), followed by SOTs (52.9%), AOTs (38.4%), and CEOTs (0%). MT staining exhibited statistically significant differences between the SABs and the SOTs (P = 0.0047) and the AOTs (P = 0.0022). A weak-to-strong positive correlation between IMT and IK or IP was observed in SABs and SOTs, whereas a strong negative correlation was observed in AOTs. No differences in IMT, IK, and IP were observed between inflammation groups A and B. The increased MT expression observed in the SABs might be correlated with clinical behavior (local invasiveness and high rate of recurrence). In the SABs and SOTs, MT plays a role in the stimulation of cellular proliferation. In contrast, MT can inhibit cellular proliferation in the AOT. The IMT, IK, and IP are not affected by inflammation.

Nucleotide precursors prevent folic acid-resistant neural tube defects in the mouse

Closure of the neural tube during embryogenesis is a crucial step in development of the central nervous system. Failure of this process results in neural tube defects, including spina bifida and anencephaly, which are among the most common birth defects worldwide. Maternal use of folic acid supplements reduces risk of neural tube defects but a proportion of cases are not preventable. Folic acid is thought to act through folate one-carbon metabolism, which transfers one-carbon units for methylation reactions and nucleotide biosynthesis. Hence suboptimal performance of the intervening reactions could limit the efficacy of folic acid. We hypothesized that direct supplementation with nucleotides, downstream of folate metabolism, has the potential to support neural tube closure. Therefore, in a mouse model that exhibits folic acid-resistant neural tube defects, we tested the effect of specific combinations of pyrimidine and purine nucleotide precursors and observed a significant protective effect. Labelling in whole embryo culture showed that nucleotides are taken up by the neurulating embryo and incorporated into genomic DNA. Furthermore, the mitotic index was elevated in neural folds and hindgut of treated embryos, consistent with a proposed mechanism of neural tube defect prevention through stimulation of cellular proliferation. These findings may provide an impetus for future investigations of supplemental nucleotides as a means to prevent a greater proportion of human neural tube defects than can be achieved by folic acid alone.

Review of the Currently Available Systems to Obtain Platelet Related Products to Treat Equine Musculoskeletal Injuries

Lameness secondary to chronic or acute injuries of the musculoskeletal system is a cause of equine wastage. This old problem has been traditionally managed with treatments, such as corticosteroids and non steroidal antiinflammatory drugs. However, recent discoveries in the pathophysiology of these diseases have provided new therapeutic approaches to their treatments. The stimulation of cellular proliferation with increase in the anabolic capacity of the resident cells for producing a good quality extracellular matrix represents a real avenue for the treatment of these diseases in horses. The injection of platelet related products has won great acceptation between equine practitioners, since these substances can be obtained from the same patient by using different procedures or devices in field conditions. These substances are source of anti-inflammatory, angiogenic and anabolic proteins (i.e., growth factors and cytokines) and cells with immunomodulatory activity (i.e., lymphocytes and macrophages). However, the cellular and molecular content of these substances depend on the method or kit used for their preparation. A review about patented and non patented technologies and procedures for the treatment of musculoskeletal diseases in horses by using platelet related products is presented in this article. Keywords: Autologous platelet concentrates, chronic musculoskeletal diseases, desmopathy, devices, growth factors, horse, osteoarthritis, platelet gel, platelet rich plasma, tendinopathy

Identification of JAK2 as a Mediator of FIP1L1-PDGFRA-Induced Eosinophil Growth and Function in CEL

The Fip1-like1 (FIP1L1)-platelet-derived growth factor receptor alpha fusion gene (F/P) arising in the pluripotent hematopoietic stem cell (HSC),causes 14% to 60% of patients with hypereosinophilia syndrome (HES). These patients, classified as having F/P (+) chronic eosinophilic leukemia (CEL), present with clonal eosinophilia and display a more aggressive disease phenotype than patients with F/P (–) HES patients. The mechanisms underlying predominant eosinophil lineage targeting and the cytotoxicity of eosinophils in this leukemia remain unclear. Given that the Janus tyrosine kinase (JAK)/signal transducers and activators of transcription (Stat) signaling pathway is key to cytokine receptor-mediated eosinophil development and activated Stat3 and Stat5 regulate the expression of genes involved in F/P malignant transformation, we investigated whether and how JAK proteins were involved in the pathogenesis of F/P-induced CEL. F/P activation of JAK2, Stat3 and Stat5, were confirmed in all the 11 F/P (+) CEL patients examined. In vitro inhibition of JAK2 in EOL-1, primary F/P(+) CEL cells (PC) and T674I F/P Imatinib resistant cells(IR) by either JAK2-specific short interfering RNA (siRNA) or the tryphostin derivative AG490(AG490), significantly reduced cellular proliferation and induced cellular apoptosis. The F/P can enhance the IL-5-induced JAK2 activation, and further results indicated that JAK2 inhibition blocked IL-5-induced cellular migration and activation of the EOL-1 and PC cells in vitro. F/P-stimulation of the JAK2 suppressed cells led to a significantly reduction in Stat3 activation, but relatively normal induction of Stat5 activation. Interestingly, JAK2 inhibition also reduced PI3K, Akt and NF-κB activity in a dose-dependent manner, and suppressed expression levels of c-Myc and Survivin. These results strongly suggest that JAK2 is activated by F/P and is required for F/P stimulation of cellular proliferation and infiltration, possibly through induction of c-Myc and Survivin expression via activation of multiple signaling pathways, including NF-κB, Stat3, and PI3K/Akt.

Epidermal Growth Factor‐Activated Extracellular Signal‐Regulated Kinase Suppresses Growth Hormone Expression and Stimulates Proliferation in MtT/ E Cells

The mechanism for the inhibition of growth hormone (GH) expression by the epidermal growth factor (EGF) was examined in two clonal cell lines, MtT/E and MtT/S. The former has a negligible basal level of GH, whereas the latter has a high basal GH. The treatment of MtT/E cells with retinoic acid resulted in a significant increase in GH mRNA and subsequently GH. This stimulatory response to retinoic acid was strongly suppressed by EGF. This suppression was associated with an increase in the phosphorylation of extracellular signal-regulated kinase 1 and 2 (Erk1/2). The MEK [mitogen-activated protein kinase (MAPK) kinases that activate ERK1 and ERK2] inhibitor, PD98059, clearly inhibited the phosphorylation of Erk1/2 and restored the stimulatory effects of retinoic acid. These results suggest that the inhibitory effects of EGF on GH expression are mediated by MAPK activation in these cells. By contrast to the GH-producing clones examined previously, EGF showed a marked stimulation of proliferation of the MtT/E cells through a mechanism dependent on MAPK activation. On the other hand, the inhibitory effect of EGF on GH expression was less pronounced and the stimulation of cellular proliferation was not seen in MtT/S cells, even though it induced Erk-phosphorylation similar to that seen in MtT/E. The distinct difference in the response to EGF between these two GH cell lines appears to be attributed to differences in the function of MAPK cascade in each cell line. This may reflect the developmental stage of the cells from which MtT/E and MtT/S are derived.

Enteric Commensal Bacteria Induce Extracellular Signal-regulated Kinase Pathway Signaling via Formyl Peptide Receptor-dependent Redox Modulation of Dual Specific Phosphatase 3

The normal microbial occupants of the mammalian intestine are crucial for maintaining gut homeostasis, yet the mechanisms by which intestinal cells perceive and respond to the microbiota are largely unknown. Intestinal epithelial contact with commensal bacteria and/or their products has been shown to activate noninflammatory signaling pathways, such as extracellular signal-related kinase (ERK), thus influencing homeostatic processes. We previously demonstrated that commensal bacteria stimulate ERK pathway activity via interaction with formyl peptide receptors (FPRs). In the current study, we expand on these findings and show that commensal bacteria initiate ERK signaling through rapid FPR-dependent reactive oxygen species (ROS) generation and subsequent modulation of MAP kinase phosphatase redox status. ROS generation induced by the commensal bacteria Lactobacillus rhamnosus GG and the FPR peptide ligand, N-formyl-Met-Leu-Phe, was abolished in the presence of selective inhibitors for G protein-coupled signaling and FPR ligand interaction. In addition, pretreatment of cells with inhibitors of ROS generation attenuated commensal bacteria-induced ERK signaling, indicating that ROS generation is required for ERK pathway activation. Bacterial colonization also led to oxidative inactivation of the redox-sensitive and ERK-specific phosphatase, DUSP3/VHR, and consequent stimulation of ERK pathway signaling. Together, these data demonstrate that commensal bacteria and their products activate ROS signaling in an FPR-dependent manner and define a mechanism by which cellular ROS influences the ERK pathway through a redox-sensitive regulatory circuit.

Application of heparinized cellulose matrices for substrate-mediated bFGF peptide and transgene delivery to stimulate cellular proliferation

We investigated the use of heparinized cellulose matrices (H-CM) as affinity substrates for binding of basic fibroblast growth factor (bFGF), a heparin-binding peptide, to facilitate cellular proliferation and substrate-mediated transgene delivery. Using human HT-1080 fibroblasts and Saos-2 osteoblasts as cellular models, we showed that H-CM was a friendly substrate for cellular adhesion. Once adhered, cells received stimulation from the bound bFGF, leading to enhanced proliferation. Furthermore, taking advantage of the negative zeta potential of H-CM, we applied electrostatic adsorption to immobilize cationic poly-ethylenimine/DNA polyplexes onto the surface for transgene delivery upon cellular adhesion. Because bFGF stimulated cellular proliferation, we observed a significant increase in transfection efficiency in comparison to transfection on H-CM without the bFGF binding. We showed that H-CM was capable of mediating both bFGF peptide and bFGF transgene delivery to induce a synergistic stimulation of cellular proliferation, thus offering a useful device for fabrication of tissue scaffolds.

Zinc Released from Injured Cells Is Acting via the Zn2+-sensing Receptor, ZnR, to Trigger Signaling Leading to Epithelial Repair

A role for Zn(2+) in accelerating wound healing is established, yet, the signaling pathways linking Zn(2+) to tissue repair are not well known. We show that in the human HaCaT keratinocytes extracellular Zn(2+) induces a metabotropic Ca(2+) response that is abolished by silencing the expression of the G-protein-coupled receptor GPR39, suggesting that this Zn(2+)-sensing receptor, ZnR, is mediating the response. Keratinocytic-ZnR signaling is highly selective for Zn(2+) and can be triggered by nanomolar concentrations of this ion. Interestingly, Zn(2+) was also released following cellular injury, as monitored by a specific non-permeable fluorescent Zn(2+) probe, ZnAF-2. Chelation of Zn(2+) and scavenging of ATP from conditioned medium, collected from injured epithelial cultures, was sufficient to eliminate the metabotropic Ca(2+) signaling. The signaling triggered by Zn(2+), via ZnR, or by ATP further activated MAP kinase and induced up-regulation of the sodium/proton exchanger NHE1 activity. Finally, activation of ZnR/GPR39 signaling or application of ATP enhanced keratinocytes scratch closure in an in vitro model. Thus our results indicate that extracellular Zn(2+), which is either applied or released following injury, activates ZnR/GPR39 to promote signaling leading to epithelial repair.

Modern proteomic methodologies for the characterization of lactosylation protein targets in milk

Heat treatment of milk induces the Maillard reaction between lactose and proteins; in this context, β-lactoglobulin and α-lactalbumin adducts have been used as markers to monitor milk quality. Since some milk proteins have been reported as essential for the delivery of microelements and, being resistant against proteolysis in the gastrointestinal tract, also contributing to the acquired immune response against pathogens and the stimulation of cellular proliferation, it is crucial to systematically determine the milk subproteome affected by the Maillard reaction for a careful evaluation of aliment functional properties. This is more important when milk is the unique nutritional source, as in infant diet. To this purpose, a combination of proteomic procedures based on analyte capture by combinatorial peptide ligand libraries, selective trapping of lactosylated peptides by m-aminophenylboronic acid-agarose chromatography and collision-induced dissociation and electron transfer dissociation MS was used for systematic identification of the lactosylated proteins in milk samples subjected to different thermal treatments. An exhaustive modification of proteins was observed in milk powdered preparations for infant nutrition. Globally, this approach allowed the identification of 271 non-redundant modification sites in 33 milk proteins, which also included low-abundance components involved in nutrient delivery, defence response against virus/microorganisms and cellular proliferative events. A comparison of the modified peptide identification percentages resulting from electron transfer dissociation or collision-induced dissociation fragmentation spectra confirmed the first activation mode as most advantageous for the analysis of lactosylated proteins. Nutritional, biological and toxicological consequences of these findings are discussed on the basis of the recent literature on this subject, emphasizing their impact on newborn diet.

Prolactin and systemic malignancies: a close association

To the editor: I read with great interest the recent article by Hietala et al. [1] about the relationship of prolactin levels and the risk of breast cancer. The results of the study by Hietala et al. clearly confirms the close association between high prolactin levels and an increased risk for breast cancer. What is often forgotten is the fact that prolactin also increases the risk for other systemic malignancies such as leukemias. For instance, Dagvadorj et al. [2] in a recent study have demonstrated that prolactin is expressed in nearly 62% of prostate cancer metastasis. In the study they also demonstrated that prolactin promotes the same proliferation of prostate cancer cells through the Jak2-Stat5a/b pathway. Endogenous cannabinoids inhibit prolactin activity and have been demonstrated to be potent inhibitors of proliferation of prostate cancer cells secondary to prolactin [3]. Similarly, in another recent study by Hatfill et al. [4], elevated prolactin levels were found in 57% of patients with acute myeloid leukemia. Prolactin has also been demonstrated to increase proliferation in T cell lymphomas [5]. Altered expression of bcl-2 and bax in lymphoma cells stimulated by prolactin may be involved in the rapid progression of these tumors [6]. Recent studies also suggest that tyrosine kinase may be involved in the transduction pathway used by prolactin for stimulation of cellular proliferation in lymphoma cells [7]. Recently, a prolactin receptor antagonist S179D prolactin has been identified that may play a major role in the near future in the management of the above mentioned tumors [8]. Wu et al. [9] in a recent study have also shown that S179D prolactin sensitizes prostate cancer cells so that even physiological concentrations of 1, 25 dihydroxy vitamin D3 result in significant apoptosis and the potent inhibition of proliferation in prostate tumors. Further studies are needed to further expand the knowledge about the role of prolactin in the etiopathogenesis of nonmammary tumors and to further identify inhibitors that may be safely and effectively employed in the management of these malignancies.

Effect of Alloxan-Diabetes on Gastrin-Releasing Peptide (GRP) Immunoreactivity in the Gastrointestinal Tract, of Sprague Dawley Rats and How This May Affect Some of the Diabetic Complications

Gastrin-releasing peptide (GRP) is the mammalian analogue of bombesin. Both neuroendocrine peptides have similar distribution, functions and immunoreactivity. The immunoreactivity for GRP has been demonstrated in the esophagus, stomach and small intestine. This peptide is reported to have various biological and pharmacological properties, which include the release of gastrointestinal hormones, control of satiety, gastrointestinal motility and stimulation of cellular proliferation which results in wound healing. It is also implicated in the proliferation of many gastrointestinal (GI), renal and prostatic tumours. Transient increase in GRP synthesis in the brain and serum concentration, which was later followed by decreased serum concentration, has been reported in hyperglycemic states. The aim of this investigation was to determine the effect of hyperglycemia on GRP secreting neurons in the sub mucosa of the GIT and how this may affect some of the GI complications of diabetes. Result showed decreased immunoreactivity to GRP in the sub mucosal neurons of the stomach and small intestine in alloxan-diabetic Sprague Dawley rats. We conclude that this could contribute to the hyperglycemia-induced GRP decrease in diabetes. This could contribute to reduced peristalsis, with resultant constipation in diabetics. It is also possible that this may also contribute to poor wound healing in diabetics.