Long-term Effects of the pituitary-adenylate cyclase-activating Polypeptide (PACAP38) in the Adult Mouse Retina: Microglial Activation and Induction of Neural Proliferation.

The degenerative retinal disorders characterized by progressive cell death and exacerbating inflammation lead ultimately to blindness. The ubiquitous neuropeptide, PACAP38 is a promising therapeutic agent as its proliferative potential and suppressive effect on microglia might enable cell replacement and attenuate inflammation, respectively. Our previous finding that PACAP38 caused a marked increase of the amacrine cells in the adult (1-year-old) mouse retina, served as a rationale of the current study. We aimed to determine the proliferating elements and the inflammatory status of the PACAP38-treated retina. Three months old mice were intravitreally injected with 100 pmol PACAP38 at 3 months intervals (3X). Retinas of 1-year-old animals were dissected and effects on cell proliferation, and expression of inflammatory regulators were analyzed. Interestingly, both mitogenic and anti-mitogenic actions were detected after PACAP38-treatment. Further analysis of the mitogenic effect revealed that proliferating cells include microglia, endothelial cells, and neurons of the ganglion cell layer but not amacrine cells. Furthermore, PACAP38 stimulated retinal microglia to polarize dominantly into M2-phenotype but also might cause subsequent angiogenesis. According to our results, PACAP38 might dampen pro-inflammatory responses and help tissue repair by reprogramming microglia into an M2 phenotype, nonetheless, with angiogenesis as a warning side effect.

Pituitary Adenylate Cyclase-Activating Polypeptide: 30 Years in Research Spotlight and 600 Million Years in Service.

Emerging from the depths of evolution, pituitary adenylate cyclase-activating polypeptide (PACAP) and its receptors (i.e., PAC1, VPAC1, VPAC2) are present in multicellular organisms from Tunicates to humans and govern a remarkable number of physiological processes. Consequently, the clinical relevance of PACAP systems spans a multifaceted palette that includes more than 40 disorders. We aimed to present the versatility of PACAP1-38 actions with a focus on three aspects: (1) when PACAP1-38 could be a cause of a malfunction, (2) when PACAP1-38 could be the cure for a malfunction, and (3) when PACAP1-38 could either improve or impair biology. PACAP1-38 is implicated in the pathophysiology of migraine and post-traumatic stress disorder whereas an outstanding protective potential has been established in ischemia and in Alzheimer's disease. Lastly, PACAP receptors could mediate opposing effects both in cancers and in inflammation. In the light of the above, the duration and concentrations of PACAP agents must be carefully set at any application to avoid unwanted consequences. An enormous amount of data accumulated since its discovery (1989) and the first clinical trials are dated in 2017. Thus in the field of PACAP research: "this is not the end, not even the beginning of the end, but maybe the end of the beginning."

The Neuroprotective Peptide PACAP1-38 Contributes to Horizontal Cell Development in Postnatal Rat Retina.

Purpose: PACAP1-38, a member of the secretin/glucagon superfamily, is expressed in the developing retina with documented neuroprotective effects. However, its function in retinal cell differentiation has yet to be elucidated. Our goals, therefore, were to identify PAC1 expressing cells morphologically, investigate the PACAP1-38 action functionally, and establish PACAP1-38 regulated events developmentally during the first postnatal week in rat retina. Methods: P1 retinal sections or whole mounts of Wistar rats were used to reveal PAC1 and calbindin immunoreactive structures. P1, P3, or P7 pups were injected intravitreally with 100 pmol PACAP1-38. Tissues were harvested 24 hours post-treatment, then processed for calbindin immunohistochemistry to determine horizontal cell number, or 6, 12, 24 hours post-treatment for real-time PCR and immunoblots to detect PCNA expression. To localize proliferating cells, anti-PCNA antibody was applied. Results: We showed various PAC1 expressing cells in RPE, NBL, and GCL in P1 retina including calbindin positive horizontal cells. We found that PACAP1-38 induced a marked cell number increase at P3 and P7 and showed upregulated cell proliferation as its mechanism; however, it was ineffective at P1. PACAP1-38 induced proliferative cells localized in the NBL, and double-marker studies demonstrated that the induced proliferative cells were horizontal cells. Conclusions: PACAP1-38 appears to act in retinal differentiation by inducing mitosis selectively in a time and cell specific manner through PAC1. The control of horizontal cell proliferation raises the novel possibilities that (1) PACAP1-38 may be a major player in retinal patterning and (2) PACAP signaling may be critical in retinoblastoma.

Metastasis blood test by flow cytometry: in vivo cancer spheroids and the role of hypoxia.

Cancer hypoxia correlates with therapeutic resistance and metastasis, suggesting that hypoxic adaptation is a critical survival advantage for cancer stem cells (CSCs). Hypoxic metabolism, however, may be a disadvantage in aerobic circulation as the extremely low incidence of metastasis-compared to the high circulating tumor-cell numbers (CTCs)-appears to suggest. As rare metastatic CSCs still survive, we searched for a mechanism that protects them from oxygen in circulation. CSCs form multicellular spheroids in vitro from virtually all cancers tested. We asked, therefore, whether cancers also form spheroids in vivo and whether circulating spheroids play a role in metastasis. We used metabolic, apoptotic and hypoxia assays, we measured aerobic barriers and calculated hypoxia vs. spheroid-size correlations. We detected metabolic/oxidative stress in spheroids, we found correlation between stem cell presence and hypoxia and we showed that the size of hypoxic spheroids is compatible with circulation. To detect spheroids in patients, we worked out a new light-scatter flow cytometry blood test and assayed 67 metastatic and control cases. We found in vivo spheroids with positive stem cell markers in cancer blood and they showed exclusive correlation with metastasis. In conclusion, our data suggest that metastatic success depends on CSC-association with in vivo spheroids. We propose that the mechanism involves a portable "micro-niche" in spheroids that may support CSC-survival/adaptation in circulation. The new assay may establish a potential early marker of metastatic progression.

Symptotic detection of chimerism: Y does it matter?

Microchimerism (MC), transplacental acquisition of allogeneic cells from the mother (maternofetal MC) or from the fetus (fetomaternal MC) has been in the focus of research recently. Amplicons using Y-chromosome specific SRY and DYS14 sequences have been used as markers to trace cells from a male fetus in the mother. The sensitivity of these markers in formaldehyde fixed paraffin embedded samples, however, is less than optimal. To study chimerism in breast cancer we took advantage of the evolutionary history of the Y chromosome and designed amplicons on gene repeats to generate additive PCR signals. The increased sensitivity detected high incidence of male chimerism in normal breast tissues. We also showed correlation with protection from cancer with unique quantitative biology. Accumulating data from biology and medicine indicate that natural chimerism is astonishingly frequent and may affect human conditions. We hypothesize that it has significant evolutionary ramifications as well.

High male chimerism in the female breast shows quantitative links with cancer.

Clinical observations suggest that pregnancy provides protection against cancer. The mechanisms involved, however, remain unclear. Fetal cells are known to enter the mother's circulation during pregnancy and establish microchimerism. We investigated if pregnancy-related embryonic/fetal stem cell integration plays a role in breast cancer. A high-sensitivity Y-chromosome assay was developed to trace male allogeneic cells (from male fetus) in females. Fixed-embedded samples (n = 206) from both normal and breast cancer patients were screened for microchimerism. The results were combined with matching clinicopathological and histological parameters and processed statistically. The results show that in our samples (182 informative) more than half of healthy women (56%) carried male cells in their breast tissue for decades (n = 68), while only one out of five in the cancer sample pool (21%) (n = 114) (odds ratio = 4.75, CI at 95% 2.34-9.69; p = 0.0001). The data support the notion that a biological link may exist between chimerism and tissue-integrity. The correlation, however, is non-linear, since male microchimerism in excess ("hyperchimerism") is also involved in cancer. The data suggest a link between hyperchimerism and HER2-type cancers, while decreased chimerism ("hypochimerism") associates with ER/PR-positive (luminal-type) breast cancers. Chimerism levels that correlate with protection appear to be non-random and share densities with the mammary progenitor components of the stem cell lineage in the breast. The results suggest that protection may involve stem/progenitor level interactions and implicate novel quantitative mechanisms in chimerism biology.

Gene expression profiling of the androgen independent prostate cancer cells demonstrates complex mechanisms mediating resistance to docetaxel.

The molecular mechanisms conferring resistance to docetaxel in prostate cancer patients remain partially understood. We generated docetaxel resistant derivatives of the androgen independent prostate cancer cell lines PC-3 and DU-145. Docetaxel rapidly induces DU-145 cell death via apoptosis and the drug resistant cells were produced by periodically exposing proliferating DU-145 cultures to small doses of docetaxel. In PC-3 cells docetaxel induces delayed cell death via mitotic catastrophe evident by profound multinucleation and formation of giant cells. Mononucleated progeny of the giant PC-3 cells shows significant resistance to docetaxel. Gene expression profiling of these docetaxel resistant PC-3 cells revealed sets of docetaxel inducible and constitutively expressed genes associated with major cancer pathways. A contradictory overlap with DU-145 docetaxel resistant cells was also found. Analyses suggested significant changes associated with apoptotic function, DNA repair, cell growth, survival and proliferation, metabolism, maintenance of cytoskeleton and extracellular matrix formation. These cellular processes often contribute to drug resistance and our study identified a set of genes managing this phenotype. Additional analyses of the drug resistant PC-3 cells using shRNA constructs determined direct relevance of Cyclin G2 to docetaxel resistance as well as prevention of multinucleation, whereas the knockdown of upregulated CYP1B1 showed no effect on either of these processes. Downregulated GBP1 was explored by ectopic overexpression and even though GBP1 has a potential to mediate resistance to docetaxel, it was not utilized in PC-3 cells. The results suggest complex combination of gene expression pattern changes that enables resistance to docetaxel while preventing death via multinucleation.

APRIN is a unique Pds5 paralog with features of a chromatin regulator in hormonal differentiation.

Activation of steroid receptors results in global changes of gene expression patterns. Recent studies showed that steroid receptors control only a portion of their target genes directly, by promoter binding. The majority of the changes are indirect, through chromatin rearrangements. The mediators that relay the hormonal signals to large-scale chromatin changes are, however, unknown. We report here that APRIN, a novel hormone-induced nuclear phosphoprotein has the characteristics of a chromatin regulator and may link endocrine pathways to chromatin. We showed earlier that APRIN is involved in the hormonal regulation of proliferative arrest in cancer cells. To investigate its function we cloned and characterized APRIN orthologs and performed homology and expression studies. APRIN is a paralog of the cohesin-associated Pds5 gene lineage and arose by gene-duplication in early vertebrates. The conservation and domain differences we found suggest, however, that APRIN acquired novel chromatin-related functions (e.g. the HMG-like domains in APRIN, the hallmarks of chromatin regulators, are absent in the Pds5 family). Our results suggest that in interphase nuclei APRIN localizes in the euchromatin/heterochromatin interface and we also identified its DNA-binding and nuclear import signal domains. The results indicate that APRIN, in addition to its Pds5 similarity, has the features and localization of a hormone-induced chromatin regulator.

The Bik BH3-only protein is induced in estrogen-starved and antiestrogen-exposed breast cancer cells and provokes apoptosis.

Evidence has been accumulating that some estrogen-dependent human breast cancers require estrogen for not only proliferation but also survival. To obtain insights into the molecular mechanisms of apoptosis of breast cancer cells subjected to estrogen starvation or exposed to antiestrogens, we characterized changes in the gene expression profile of MCF-7/BUS human breast cancer cells and revealed a strong induction of Bik, a member of the BH3-only proapoptotic proteins. The Bik mRNA transcript and protein were strongly induced by estrogen starvation or exposure to fulvestrant, a pure antiestrogen that competes with the natural estrogens for binding to the estrogen receptors. This Bik induction preceded apoptotic cell death, which was blocked by zVAD-fmk, a pancaspase inhibitor. Amounts of the Bcl-2-related proteins, such as Bcl-2, Bcl-XL, or Bax, showed only marginal changes in the presence or absence of estrogens or antiestrogens. Suppression of Bik expression by using the small interfering RNA effectively blocked the fulvestrant-induced breast cancer cell apoptosis. These results indicate that Bik is induced in MCF-7/BUS cells in the absence of estrogen signaling and plays a critical role in the antiestrogen-provoked breast cancer cell apoptosis.

Mechanism of androgen action on cell proliferation: AS3 protein as a mediator of proliferative arrest in the rat prostate.

Androgens control the proliferation of their target cells first by increasing cell proliferation and later by inhibiting the proliferation of those same cells. Recently, we reported that the AS3 protein mediates the androgen-induced quiescence in androgen-target human cell lines. Our aims were to investigate the expression of the AS3 protein in the rat prostate in situ and in human cells in culture. Adult rats were separated into four groups (intact, castrated, castrated plus 3-d testosterone propionate replacement, and castrated plus 7-d testosterone propionate replacement). S9 cells expressing a tetracycline-regulated sense AS3 were also used. AS3 was expressed in the nuclei of over 90% of the epithelial cells and about 40% of the smooth muscle cells of the intact rat prostate. AS3 was not expressed in castrated rats or during the proliferative phase of androgen-induced regeneration. It was expressed in intact and castrated animals when the prostate has reached adult organ size. The AS3 protein was not expressed in cells that incorporate bromodeoxyuridine. These data suggest that AS3 is a mediator of the proliferative arrest in the normal rat prostate in situ and human prostate cell lines and that its expression is androgen-induced.