Rescue of p53 functions by in vitro-transcribed mRNA impedes the growth of high-grade serous ovarian cancer.

BACKGROUND: The cellular tumor protein p53 (TP53) is a tumor suppressor gene that is frequently mutated in human cancers. Among various cancer types, the very aggressive high-grade serous ovarian carcinoma (HGSOC) exhibits the highest prevalence of TP53 mutations, present in >96% of cases. Despite intensive efforts to reactivate p53, no clinical drug has been approved to rescue p53 function. In this study, our primary objective was to administer in vitro-transcribed (IVT) wild-type (WT) p53-mRNA to HGSOC cell lines, primary cells, and orthotopic mouse models, with the aim of exploring its impact on inhibiting tumor growth and dissemination, both in vitro and in vivo. METHODS: To restore the activity of p53, WT p53 was exogenously expressed in HGSOC cell lines using a mammalian vector system. Moreover, IVT WT p53 mRNA was delivered into different HGSOC model systems (primary cells and patient-derived organoids) using liposomes and studied for proliferation, cell cycle progression, apoptosis, colony formation, and chromosomal instability. Transcriptomic alterations induced by p53 mRNA were analyzed using RNA sequencing in OVCAR-8 and primary HGSOC cells, followed by ingenuity pathway analysis. In vivo effects on tumor growth and metastasis were studied using orthotopic xenografts and metastatic intraperitoneal mouse models. RESULTS: Reactivation of the TP53 tumor suppressor gene was explored in different HGSOC model systems using newly designed IVT mRNA-based methods. The introduction of WT p53 mRNA triggered dose-dependent apoptosis, cell cycle arrest, and potent long-lasting inhibition of HGSOC cell proliferation. Transcriptome analysis of OVCAR-8 cells upon mRNA-based p53 reactivation revealed significant alterations in gene expression related to p53 signaling, such as apoptosis, cell cycle regulation, and DNA damage. Restoring p53 function concurrently reduces chromosomal instability within the HGSOC cells, underscoring its crucial contribution in safeguarding genomic integrity by moderating the baseline occurrence of double-strand breaks arising from replication stress. Furthermore, in various mouse models, treatment with p53 mRNA reduced tumor growth and inhibited tumor cell dissemination in the peritoneal cavity in a dose-dependent manner. CONCLUSIONS: The IVT mRNA-based reactivation of p53 holds promise as a potential therapeutic strategy for HGSOC, providing valuable insights into the molecular mechanisms underlying p53 function and its relevance in ovarian cancer treatment.

Quantitative and qualitative evaluation of the hippocampal cytoarchitecture in adult cats with regard to the pathological diagnosis of hippocampal sclerosis.

Cats are known to be affected by hippocampal sclerosis, potentially causing antiseizure drug(s) resistance. In order to lay the foundation for a standardized, systematic classification and diagnosis of this pathology in cats, this prospective study aimed at evaluating normal reference values of cellular densities and the cytoarchitecture of the feline hippocampus. Three transverse sections (head, body and tail) of each left hippocampus were obtained from 17 non-epileptic cats of different brachycephalic and mesocephalic breeds and age classes (range: 3-17 years). Histological (hematoxylin and eosin, Nissl) and immunohistochemical (NeuN, GFAP) staining was performed to investigate neuron and astroglial cell populations, as well as the layer thickness of the pyramidal cell layer and granule cell layer. Significant differences in neuronal density (in CA2-CA4 and the granule cell layer) and layer thickness (in CA1-CA3 and the granule cell layer) were evidenced throughout the longitudinal hippocampal axis (p<0.05); on the other hand, the astrocyte density did not differ. Moreover, reference ranges were defined for these parameters in the pyramidal cell layer and in the granule cell layer. The findings did not differ according to breed or age. In veterinary medicine these parameters have not been evaluated in cats so far. As surgical treatment may become a therapeutic option for cats with temporal lobe epilepsy, estimating normal values of the hippocampal cytoarchitecture will help in the standardized histopathological examination of resected hippocampal specimens to reach a diagnosis of hippocampal sclerosis.

Modelling the Functions of Polo-Like Kinases in Mice and Their Applications as Cancer Targets with a Special Focus on Ovarian Cancer.

Polo-like kinases (PLKs) belong to a five-membered family of highly conserved serine/threonine kinases (PLK1-5) that play differentiated and essential roles as key mitotic kinases and cell cycle regulators and with this in proliferation and cellular growth. Besides, evidence is accumulating for complex and vital non-mitotic functions of PLKs. Dysregulation of PLKs is widely associated with tumorigenesis and by this, PLKs have gained increasing significance as attractive targets in cancer with diagnostic, prognostic and therapeutic potential. PLK1 has proved to have strong clinical relevance as it was found to be over-expressed in different cancer types and linked to poor patient prognosis. Targeting the diverse functions of PLKs (tumor suppressor, oncogenic) are currently at the center of numerous investigations in particular with the inhibition of PLK1 and PLK4, respectively in multiple cancer trials. Functions of PLKs and the effects of their inhibition have been extensively studied in cancer cell culture models but information is rare on how these drugs affect benign tissues and organs. As a step further towards clinical application as cancer targets, mouse models therefore play a central role. Modelling PLK function in animal models, e.g., by gene disruption or by treatment with small molecule PLK inhibitors offers promising possibilities to unveil the biological significance of PLKs in cancer maintenance and progression and give important information on PLKs' applicability as cancer targets. In this review we aim at summarizing the approaches of modelling PLK function in mice so far with a special glimpse on the significance of PLKs in ovarian cancer and of orthotopic cancer models used in this fatal malignancy.

On the origin of germ cell neoplasia in situ: Dedifferentiation of human adult Sertoli cells in cross talk with seminoma cells in vitro.

Germ cell neoplasia in situ (GCNIS) is the noninvasive precursor of testicular germ cell tumors type II, the most common cancer in young men, which originates from embryonic germ cells blocked in their maturation. GCNIS is associated with impaired Sertoli cells (SCs) that express fetal keratin 18 (KRT18) and the pluripotency factor SRY-Box transcription factor 2 (SOX2). According to the current theory concerning the origin of GCNIS, these SCs are prepubertal cells arrested in their maturation due to (epi)genetic anomalies and/or environmental antiandrogens. Thus, they are unable to support the development of germ cells, which leads to their maturational block and further progresses into GCNIS. Alternatively, these SCs are hypothesized to be adult cells dedifferentiating secondarily under the influence of GCNIS. To examine whether tumor cells can dedifferentiate SCs, we established a coculture model of adult human SCs (FS1) and a seminoma cell line similar to GCNIS (TCam-2). After 2 wk of coculture, FS1 cells showed progressive expression of KRT18 and SOX2, mimicking the in vivo changes. TCam-2 cells showed SOX2 expression and upregulation of further pluripotency- and reprogramming-associated genes, suggesting a seminoma to embryonal carcinoma transition. Thus, our FS1/TCam-2 coculture model is a valuable tool for investigating interactions between SCs and seminoma cells. Our immunohistochemical and ultrastructural studies of human testicular biopsies with varying degrees of GCNIS compared to biopsies from fetuses, patients with androgen insensitivity syndrome, and patients showing normal spermatogenesis further suggest that GCNIS-associated SCs represent adult cells undergoing progressive dedifferentiation.

Partial cortico-hippocampectomy in cats, as therapy for refractory temporal epilepsy: A descriptive cadaveric study.

Cats, similar to humans, are known to be affected by hippocampal sclerosis (HS), potentially causing antiepileptic drug (AED) resistance. HS can occur as a consequence of chronic seizure activity, trauma, inflammation, or even as a primary disease. In humans, temporal lobe resection is the standardized therapy in patients with refractory temporal lobe epilepsy (TLE). The majority of TLE patients are seizure free after surgery. Therefore, the purpose of this prospective cadaveric study is to establish a surgical technique for hippocampal resection in cats as a treatment for AED resistant seizures. Ten cats of different head morphology were examined. Pre-surgical magnetic resonance imaging (MRI) and computed tomography (CT) studies of the animals' head were carried out to complete 3D reconstruction of the head, brain, and hippocampus. The resected hippocampal specimens and the brains were histologically examined for tissue injury adjacent to the hippocampus. The feasibility of the procedure, as well as the usability of the removed specimen for histopathological examination, was assessed. Moreover, a micro-CT (mCT) examination of the brain of two additional cats was performed in order to assess temporal vasculature as a reason for possible intraoperative complications. In all cats but one, the resection of the temporal cortex and the hippocampus were successful without any evidence of traumatic or vascular lesions in the surrounding neurovascular structures. In one cat, the presence of mechanical damage (a fissure) of the thalamic surface was evident in the histopathologic examination of the brain post-resection. All hippocampal fields and the dentate gyrus were identified in the majority of the cats via histological examination. The study describes a new surgical approach (partial temporal cortico-hippocampectomy) offering a potential treatment for cats with clinical and diagnostic evidence of temporal epilepsy which do not respond adequately to the medical therapy.

Cell behavior of human mesenchymal stromal cells in response to silica/collagen based xerogels and calcium deficient culture conditions.

Herein, we aim to elucidate osteogenic effects of two silica-based xerogels with different degrees of bioactivity on human bone-derived mesenchymal stromal cells by means of scanning electron microscopy, quantitative PCR enhanced osteogenic effects and the formation of an extracellular matrix which could be ascribed to the sample with lower bioactivity. Given the high levels of bioactivity, the cells revealed remarkable sensitivity to extremely low calcium levels of the media. Therefore, additional experiments were performed to elucidate cell behavior under calcium deficient conditions. The results refer to capacity of the bone-derived stromal cells to overcome calcium deficiency even though proliferation, migration and osteogenic differentiation capabilities were diminished. One reason for the differences of the cellular response (on tissue culture plates versus xerogels) to calcium deficiency seems to be the positive effect of silica. The silica could be detected intracellularly as shown by time of flight-secondary ion mass spectrometry after cultivation of primary cells for 21 days on the surfaces of the xerogels. Thus, the present findings refer to different osteogenic differentiation potentials of the xerogels according to the different degrees of bioactivity, and to the role of silica as a stimulator of osteogenesis. Finally, the observed pattern of connexin-based hemichannel gating supports the assumption that connexin 43 is a key factor for calcium-mediated osteogenesis in bone-derived mesenchymal stromal cells.

Re-visiting the Protamine-2 locus: deletion, but not haploinsufficiency, renders male mice infertile.

Protamines are arginine-rich DNA-binding proteins that replace histones in elongating spermatids. This leads to hypercondensation of chromatin and ensures physiological sperm morphology, thereby protecting DNA integrity. In mice and humans, two protamines, protamine-1 (Prm1) and protamine-2 (Prm2) are expressed in a species-specific ratio. In humans, alterations of this PRM1/PRM2 ratio is associated with subfertility. By applying CRISPR/Cas9 mediated gene-editing in oocytes, we established Prm2-deficient mice. Surprisingly, heterozygous males remained fertile with sperm displaying normal head morphology and motility. In Prm2-deficient sperm, however, DNA-hypercondensation and acrosome formation was severely impaired. Further, the sperm displayed severe membrane defects resulting in immotility. Thus, lack of Prm2 leads not only to impaired histone to protamine exchange and disturbed DNA-hypercondensation, but also to severe membrane defects resulting in immotility. Interestingly, previous attempts using a regular gene-targeting approach failed to establish Prm2-deficient mice. This was due to the fact that already chimeric animals generated with Prm2+/- ES cells were sterile. However, the Prm2-deficient mouse lines established here clearly demonstrate that mice tolerate loss of one Prm2 allele. As such they present an ideal model for further studies on protamine function and chromatin organization in murine sperm.

Specific localisation of gap junction protein connexin 32 in the gastric mucosa of horses.

In the glandular stomach, gap junctional intercellular communication (GJIC) plays an important role in the gastric mucosal defense system, and loss of GJIC is associated with ulcer formation. In spite of the high incidence of gastric ulcers in horses, particularly at pars nonglandularis, the presence of gap junctions in the equine stomach has not yet been studied. The objective was to obtain basic data on the distribution of gap junction protein connexin 32 (Cx32) in the different regions of normal equine gastric mucosa. Samples of mucosa were taken from seven horses at cardiac, fundic, and pyloric region and pars nonglandularis. To detect Cx32, immunohistochemical staining and Western blot analysis were performed. Corresponding mRNA was shown by RT-PCR and localised in tissue sections by in situ hybridisation. Cx32 was found in the glandular regions, whereas it was not detectable in squamous mucosa. Within the glandular epithelium, Cx32 was abundant in surface and foveolar cells and decreased towards the proliferative zone of the glands. These results suggest that gap junctions develop during the maturation of surface cells. Whether the lack of Cx32 at pars nonglandularis contributes to its susceptibility for developing ulcers, has to be further elucidated.