Cancer-Associated Fibroblasts in Pancreatic Ductal Adenocarcinoma or a Metaphor for Heterogeneity: From Single-Cell Analysis to Whole-Body Imaging.

Pancreatic ductal adenocarcinoma (PDAC) represents a formidable challenge due to its aggressive nature and poor prognosis. The tumor microenvironment (TME) in PDAC, characterized by intense stromal desmoplastic reactions and a dominant presence of cancer-associated fibroblasts (CAFs), significantly contributes to therapeutic resistance. However, within the heterogeneous CAF population, fibroblast activation protein (FAP) emerges as a promising target for Gallium-68 FAP inhibitor positron emission tomography (Ga68FAPI-PET) imaging. Notably, 68Ga-FAPI-PET demonstrates promising diagnostic sensitivity and specificity, especially in conjunction with low tracer uptake in non-tumoral tissues. Moreover, it provides valuable insights into tumor-stroma interactions, a critical aspect of PDAC tumorigenesis not adequately visualized through conventional methods. The clinical implications of this innovative imaging modality extend to its potential to reshape treatment strategies by offering a deeper understanding of the dynamic TME. However, while the potential of 68Ga-FAPI-PET is evident, ongoing correlative studies are essential to elucidate the full spectrum of CAF heterogeneity and to validate its impact on PDAC management. This article provides a comprehensive review of CAF heterogeneity in PDAC and explores the potential impact of 68Ga-FAPI-PET on disease management.

High-resolution and quantitative spatial analysis reveal intra-ductal phenotypic and functional diversification in pancreatic cancer.

A 'classical' and a 'basal-like' subtype of pancreatic cancer have been reported, with differential expression of GATA6 and different dosages of mutant KRAS. We established in situ detection of KRAS point mutations and mRNA panels for the consensus subtypes aiming to project these findings to paraffin-embedded clinical tumour samples for spatial quantitative analysis. We unveiled that, next to inter-patient and intra-patient inter-ductal heterogeneity, intraductal spatial phenotypes exist with anti-correlating expression levels of GATA6 and KRASG12D . The basal-like mRNA panel better captured the basal-like cell states than widely used protein markers. The panels corroborated the co-existence of the classical and basal-like cell states in a single tumour duct with functional diversification, i.e. proliferation and epithelial-to-mesenchymal transition respectively. Mutant KRASG12D detection ascertained an epithelial origin of vimentin-positive cells in the tumour. Uneven spatial distribution of cancer-associated fibroblasts could recreate similar intra-organoid diversification. This extensive heterogeneity with functional cooperation of plastic tumour cells poses extra challenges to therapeutic approaches. © 2023 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of The Pathological Society of Great Britain and Ireland.

Resistance to Gemcitabine in Pancreatic Cancer Is Connected to Methylglyoxal Stress and Heat Shock Response.

Pancreatic ductal adenocarcinoma (PDAC) is a fatal disease with poor prognosis. Gemcitabine is the first-line therapy for PDAC, but gemcitabine resistance is a major impediment to achieving satisfactory clinical outcomes. This study investigated whether methylglyoxal (MG), an oncometabolite spontaneously formed as a by-product of glycolysis, notably favors PDAC resistance to gemcitabine. We observed that human PDAC tumors expressing elevated levels of glycolytic enzymes together with high levels of glyoxalase 1 (GLO1), the major MG-detoxifying enzyme, present with a poor prognosis. Next, we showed that glycolysis and subsequent MG stress are triggered in PDAC cells rendered resistant to gemcitabine when compared with parental cells. In fact, acquired resistance, following short and long-term gemcitabine challenges, correlated with the upregulation of GLUT1, LDHA, GLO1, and the accumulation of MG protein adducts. We showed that MG-mediated activation of heat shock response is, at least in part, the molecular mechanism underlying survival in gemcitabine-treated PDAC cells. This novel adverse effect of gemcitabine, i.e., induction of MG stress and HSR activation, is efficiently reversed using potent MG scavengers such as metformin and aminoguanidine. We propose that the MG blockade could be exploited to resensitize resistant PDAC tumors and to improve patient outcomes using gemcitabine therapy.

What did COVID-19 pandemics teach us about single-fraction radiotherapy for painful bone metastases-State of the art or undertreatment?

BACKGROUND: Choosing the optimal treatment approach for patients with painful bone metastases during the COVID-19 pandemic became challenging. A simple technique, single fraction radiotherapy was recommended for these patients usually referring to bone metastases as a single entity, although it is a very heterogeneous group of patients. AIM: This study aimed to analyze the response to palliative single fraction radiotherapy in relation to age, performance status, primary tumor, histopathology, and bone localization in the group of patients with painful bone metastases. METHODS: A clinical, prospective, non-randomized study was conducted at the Institute for Oncology and Radiology of Serbia, which included 64 patients with noncomplicated, painful bone metastases who underwent palliative, pain-relieving radiation therapy with a single tumor dose of 8Gy in a single hospital visit. Response to treatment was patient reported via telephone interview using visual analog scale. The response assessment was based on the international consensus panel of radiation oncologists. RESULTS: In the entire group of patients, 83% responded to radiotherapy. No statistically significant difference was observed in response to therapy, time to reach the maximum response, degree of pain reduction, nor in response duration depending on the patient's age, performance status, the primary origin of the tumor, histopathology, or location of the metastasis (bone) that was irradiated. CONCLUSION: Regardless of clinical parameters, palliative radiotherapy with a single dose of 8Gy can be considered very effective in quick pain relief in patients with noncomplicated painful bone metastases. Single fraction radiotherapy in a single hospital visit, as well as patient-reported outcome for these patients may be considered favorable beyond Covid pandemics.

Combination, Modulation and Interplay of Modern Radiotherapy with the Tumor Microenvironment and Targeted Therapies in Pancreatic Cancer: Which Candidates to Boost Radiotherapy?

Pancreatic ductal adenocarcinoma cancer (PDAC) is a highly diverse disease with low tumor immunogenicity. PDAC is also one of the deadliest solid tumor and will remain a common cause of cancer death in the future. Treatment options are limited, and tumors frequently develop resistance to current treatment modalities. Since PDAC patients do not respond well to immune checkpoint inhibitors (ICIs), novel methods for overcoming resistance are being explored. Compared to other solid tumors, the PDAC's tumor microenvironment (TME) is unique and complex and prevents systemic agents from effectively penetrating and killing tumor cells. Radiotherapy (RT) has the potential to modulate the TME (e.g., by exposing tumor-specific antigens, recruiting, and infiltrating immune cells) and, therefore, enhance the effectiveness of targeted systemic therapies. Interestingly, combining ICI with RT and/or chemotherapy has yielded promising preclinical results which were not successful when translated into clinical trials. In this context, current standards of care need to be challenged and transformed with modern treatment techniques and novel therapeutic combinations. One way to reconcile these findings is to abandon the concept that the TME is a well-compartmented population with spatial, temporal, physical, and chemical elements acting independently. This review will focus on the most interesting advancements of RT and describe the main components of the TME and their known modulation after RT in PDAC. Furthermore, we will provide a summary of current clinical data for combinations of RT/targeted therapy (tRT) and give an overview of the most promising future directions.

CDK4/6 Inhibitors in Pancreatobiliary Cancers: Opportunities and Challenges.

Existing treatment strategies for pancreatobiliary malignancies are limited. Nowadays, surgery is the only path to cure these types of cancer, but only a small number of patients present with resectable tumors at the time of diagnosis. The notoriously poor prognosis, lack of diverse treatment options associated with pancreaticobiliary cancers, and their resistance to current therapies reflect the urge for the development of novel therapeutic targets. Cyclin-dependent kinase 4/6 (CDK4/6) inhibitors have emerged as an attractive therapeutic strategy in a number of cancers since their approval for treatment in patients with ER+/HER- breast cancer in combination with antiestrogens. In this article, we discuss the therapeutic potential of CDK4/6 inhibitors in pancreatobiliary cancers, notably cholangiocarcinoma and pancreatic ductal adenocarcinoma.

A Comprehensive Review of the Potential Role of Liquid Biopsy as a Diagnostic, Prognostic, and Predictive Biomarker in Pancreatic Ductal Adenocarcinoma.

Pancreatic ductal adenocarcinoma is one of the most lethal malignant diseases, with a mortality rate being close to incidence. Due to its heterogeneity and plasticity, as well as the lack of distinct symptoms in the early phases, it is very often diagnosed at an advanced stage, resulting in poor prognosis. Traditional tissue biopsies remain the gold standard for making a diagnosis, but have an obvious disadvantage in their inapplicability for frequent sampling. Blood-based biopsies represent a non-invasive method which potentially offers easy and repeated sampling, leading to the early detection and real-time monitoring of the disease and hopefully an accurate prognosis. Given the urgent need for a reliable biomarker that can estimate a patient's condition and response to an assigned treatment, blood-based biopsies are emerging as a potential new tool for improving patients' survival and surveillance. In this article, we discuss the current advances and challenges in using liquid biopsies for pancreatic cancer, focusing on circulating tumour DNA (ctDNA), extracellular vesicles (EVs), and circulating tumour cells (CTCs), and compare the performance and reliability of different biomarkers and combinations of biomarkers.

Association between microRNAs 10b/21/34a and acute toxicity in glioblastoma patients treated with radiotherapy and temozolomide.

A personalized approach to chemoradiation is important in reducing its potential side effects and identifying a group of patients prone to toxicity. MicroRNAs have been shown to have a predictive potential for radiotoxicity. The goal of the study was to test if levels of miRNA in peripheral blood mononuclear cells of glioblastoma patients are associated with toxicity and to identify the peak time point for toxicity. MicroRNA-10b/21/34a levels were measured in 43 patients with and without toxicity, at baseline, at the 15th, and at the 30th fraction by Real-Time quantitative Polymerase Chain Reaction. MicroRNA-10b/21 levels increased with toxicity grade (p = 0.014; p = 0.013); miR-21/34a levels were significantly different between patients with and without toxicity at the 15th fraction (p = 0.030; p = 0.045), while miR-34a levels significantly changed during treatment (p < 0.001). All three miRNAs showed a significantly high positive correlation with one another. MiR-34a might be considered as a predictive factor for toxicity due to its changes during treatment, and differences between the groups with and without toxicity; miR-10b might be used to predict toxicity; miR-10b/21 might be used for predicting the grade of toxicity in GB patients.

Clinical analysis of COVID-19 positive cancer inpatients in National Cancer Center in Serbia.

INTRODUCTION: The outbreak of COVID-19 has had an impact on global healthcare as well as on radiotherapy practice in many countries. This study aimed to identify clinical characteristics of Coronavirus Disease 2019 (COVID-19) infected cancer inpatients, as well as what impact this infection had on radiation treatment of the patients. METHODOLOGY: In this retrospective study, we included cancer inpatients with laboratory confirmed COVID-19 infection during the radiotherapy or chemoradiation in April 2020 in National Cancer Research Center in Serbia. Data were obtained from the medical records between 1 April and 1 July 2020. RESULTS: A total of 49 COVID-19 infected cancer inpatients were included. The most frequently reported cancers were head and neck cancers, in twenty-three patients (46.8%). Lymphopenia was present in 77.5% of the patients. Red blood cells, haemoglobin and platelets were significantly lower during incubation or diagnosis of COVID-19. Twenty-seven (55.1%) patients did not finish radiotherapy. The age of patients who finished radiotherapy after COVID-19 infection was significantly lower compared to the patients who did not finish radiotherapy (60.5 ± 7.8 vs. 68.6 ± 11.2; p < 0.005). CONCLUSIONS: COVID-19 infected cancer patients in radiotherapy practice show similar symptoms and demographic characteristics as the general population infected with SARS-CoV-2 virus. Patients with head and neck cancers may be susceptible to infection with COVID-19. Old age and male gender may be risk factors for discontinuation of radiotherapy in COVID-19 infected cancer patients.

Discovery and 3D imaging of a novel ΔNp63-expressing basal cell type in human pancreatic ducts with implications in disease.

OBJECTIVE: The aggressive basal-like molecular subtype of pancreatic ductal adenocarcinoma (PDAC) harbours a ΔNp63 (p40) gene expression signature reminiscent of a basal cell type. Distinct from other epithelia with basal tumours, ΔNp63+ basal cells reportedly do not exist in the normal pancreas. DESIGN: We evaluated ΔNp63 expression in human pancreas, chronic pancreatitis (CP) and PDAC. We further studied in depth the non-cancerous tissue and developed a three-dimensional (3D) imaging protocol (FLIP-IT, Fluorescence Light sheet microscopic Imaging of Paraffin-embedded or Intact Tissue) to study formalin-fixed paraffin-embedded samples at single cell resolution. Pertinent mouse models and HPDE cells were analysed. RESULTS: In normal human pancreas, rare ΔNp63+ cells exist in ducts while their prevalence increases in CP and in a subset of PDAC. In non-cancer tissue, ΔNp63+ cells are atypical KRT19+ duct cells that overall lack SOX9 expression while they do express canonical basal markers and pertain to a niche of cells expressing gastrointestinal stem cell markers. 3D views show that the basal cells anchor on the basal membrane of normal medium to large ducts while in CP they exist in multilayer dome-like structures. In mice, ΔNp63 is not found in adult pancreas nor in selected models of CP or PDAC, but it is induced in organoids from larger Sox9low ducts. In HPDE, ΔNp63 supports a basal cell phenotype at the expense of a classical duct cell differentiation programme. CONCLUSION: In larger human pancreatic ducts, basal cells exist. ΔNp63 suppresses duct cell identity. These cells may play an important role in pancreatic disease, including PDAC ontogeny, but are not present in mouse models.

Pancreatic Cancer Meets Human Microbiota: Close Encounters of the Third Kind.

Pancreatic ductal adenocarcinoma (PDAC) remains one of the most lethal types of cancer with a dismal prognosis. The five-year survival rate has not changed significantly in over 40 years. Current first-line treatments only offer a modest increase in overall survival in unselected populations, and there is an urgent need to personalize treatment in this aggressive disease and develop new therapeutic strategies. Evolving evidence suggests that the human microbiome impacts cancerogenesis and cancer resistance to therapy. The mechanism of action and interaction of microbiome and PDAC is still under investigation. Direct and indirect effects have been proposed, and the use of several microbiome signatures as predictive and prognostic biomarkers for pancreatic cancer are opening new therapeutic horizons. In this review, we provide an overview for the clinicians of studies describing the influence and associations of oral, gastrointestinal and intratumoral microbiota on PDAC development, progression and resistance to therapy and the potential use of microbiota as a diagnostic, prognostic and predictive biomarker for PDAC.

Tumour and stroma RNA signatures predict more accurately distant recurrence than clinicopathological factors in resected pancreatic adenocarcinoma.

BACKGROUND: Few patients with pancreatic adenocarcinoma (PAC) are eligible for surgery. Patients with early relapse have a poor prognosis and might be better candidates for a medical approach. Clinical and pathological parameters only partially predict recurrence and are only obtained after surgery. PAC subtypes based on gene expression were proposed, and we assessed if they could predict the risk and type of recurrence independently of clinicopathological parameters. METHODS: Patients with curative-intent surgery for PAC without pretreatment were selected and divided into two independent cohorts defined as discovery (n = 381) and validation (n = 149) cohorts. Transcriptomic analyses were performed on formalin-fixed paraffin-embedded surgical samples to characterise tumour and stroma compartments using previously defined signatures. We associated molecular and clinicopathological characteristics with general, distant, and local recurrences using Cox regression analyses. RESULTS: We found that tumour biology predicted distant recurrence contrary to local recurrence, which was directly related to resection margin status. Pure basal-like and stroma-activated subtypes were strongly associated with distant recurrence, independently of clinicopathological factors (hazard ratios [HRs] = 5.85, p < 0.001 and HR = 1.75, p = 0.007, respectively). By dissecting tumoural and stromal compartments, we demonstrated that the basal-like tumour component positively correlated with distant recurrence in both cohorts (HR = 1.45, p < 0.001 and HR = 1.90, p < 0.001), whereas the inactive structural stroma component was protective against distant recurrence (HR = 0.68, p < 0.001 and HR = 0.72, p < 0.001). CONCLUSIONS: In addition to suggesting a different mechanism for local and distant relapse (incomplete resection and high metastatic potential, respectively), our results show the potency of molecular phenotype to predict patient outcome regarding distant recurrences.

Aquaporins Involvement in Pancreas Physiology and in Pancreatic Diseases.

Aquaporins are a family of transmembrane proteins permeable to water. In mammals, they are subdivided into classical aquaporins that are permeable to water; aquaglyceroporins that are permeable to water, glycerol and urea; peroxiporins that facilitate the diffusion of H2O2 through cell membranes; and so called unorthodox aquaporins. Aquaporins ensure important physiological functions in both exocrine and endocrine pancreas. Indeed, they are involved in pancreatic fluid secretion and insulin secretion. Modification of aquaporin expression and/or subcellular localization may be involved in the pathogenesis of pancreatic insufficiencies, diabetes and pancreatic cancer. Aquaporins may represent useful drug targets for the treatment of pathophysiological conditions affecting pancreatic function, and/or diagnostic/predictive biomarker for pancreatic cancer. This review summarizes the current knowledge related to the involvement of aquaporins in the pancreas physiology and physiopathology.

Curative intent for unresectable advanced squamous cell esophageal cancer: Overall survival after chemoradiation.

PURPOSE: To analyse the overall survival (OS) of patients with locally advanced, unresectable esophageal cancer treated with chemoradiation (CRT) with or without surgery. METHODS: CRT was administered to 63 patients with locally advanced (T3-4, N0-1), initially unresectable squamous cell esophageal cancer. After the assessment of tumor response to treatment, medically fit patients converted to operable stage were subjected to surgery. Regular follow-up was performed every 3 months during first 2 years, and then every 6 months. RESULTS: All 63 patients completed the whole radiotherapy course. Forty patients (63%) received complete 4 cycles of chemotherapy. In the remaining 23 patients (37%) chemotherapy was interrupted due to toxicity. Clinical response to CRT was: complete response (CR) in 4 patients (6%), partial response (PR in 27 (43%), stable disease (SD) in 22 (35%) patients, and 10 patients (16%) had disease progression (PD). After reevaluation, 23 patients (15 PR and 8 SD after CRT) underwent surgery (37%), all with R0 resection. OS in the whole group was 53% at one year, and 36% at two years. OS was significantly better in the operated group of patients than in the non-operated group. No statistically significant difference in OS was observed comparing operated to CR patients with no surgery (70 vs 50%). In the non-operated group of patients there was no difference in OS between CR, PR, and SD patients. CONCLUSIONS: With appropriate selection, patients with advanced squamous cell esophageal cancer should be considered for potentially effective treatment.

Forskolin Inhibits Lipopolysaccharide-Induced Modulation of MCP-1 and GPR120 in 3T3-L1 Adipocytes through an Inhibition of NFκB.

In an obese state, Toll-like receptor-4 (TLR-4) upregulates proinflammatory adipokines secretion including monocyte chemotactic protein-1 (MCP-1) in adipose tissue. In contrast, G-protein coupled receptor 120 (GPR120) mediates antiobesity effects. The aim of this study was to determine the signaling pathway by which Forskolin (FK), a cyclic adenosine monophosphate- (cAMP-) promoting agent causing positive changes in body composition in overweight and obese adult men, affects MCP-1 and GPR120 expression during an inflammatory response induced by lipopolysaccharide (LPS) in adipocytes, such as in an obese state. 3T3-L1 cells differentiated into adipocytes (DC) were stimulated with LPS in the absence or presence of FK and inhibitors of TLR-4 and inhibitor of kappa B (IκBα). In DC, LPS increased MCP-1, TLR-4, and nuclear factor-κB1 (NFκB1) mRNA levels, whereas it decreased GPR120 mRNA levels. In DC, FK inhibited the LPS-induced increase in MCP-1, TLR-4, and NFκB1 mRNA levels and the LPS-induced decrease in GPR120 mRNA. BAY11-7082 and CLI-095 abolished these LPS-induced effects. In conclusion, FK inhibits LPS-induced increase in MCP-1 mRNA levels and decrease in GPR120 mRNA levels in adipocytes and may be a potential treatment for inflammation in obesity. Furthermore, TLR-4-induced activation of NFκB may be involved in the LPS-induced regulation of these genes.

Involvement of JNK/NFκB Signaling Pathways in the Lipopolysaccharide-Induced Modulation of Aquaglyceroporin Expression in 3T3-L1 Cells Differentiated into Adipocytes.

Aquaglyceroporins, belonging to the family of aquaporins (AQPs), are integral plasma membrane proteins permeable to water and glycerol that have emerged as key players in obesity. The aim of this study was to investigate the expression profile of AQPs in undifferentiated and differentiated 3T3-L1 cells and to investigate the changes in expression of aquaglyceroporins in 3T3-L1 cells differentiated into adipocytes and subjected to lipopolysaccharide (LPS) mimicking inflammation occurring during obesity. Furthermore, the study aimed at identifying the signaling cascade involved in the regulation of aquaglyceroporins expression upon LPS stimulation. 3T3-L1 cells were grown as undifferentiated cells (UDC; preadipocytes) or cells differentiated into adipocytes (DC, adipocytes). DC were incubated in the presence or absence of LPS with or without inhibitors of various protein kinases. AQPs mRNA expression levels were measured by real-time quantitative polymerase chain reaction (RT-qPCR). AQP1, AQP2, AQP3, AQP9 and AQP11 mRNA were expressed in both UDC and DC, whereas AQP4, AQP7 and AQP8 mRNA were expressed only in DC. In DC, LPS up-regulated AQP3 mRNA levels (p < 0.05) compared to control; these effects were inhibited by CLI095, SP600125 and BAY11-7082 (p < 0.05). LPS decreased both AQP7 and AQP11 mRNA levels (p < 0.01) in DC as compared to control; this decrease was inhibited by CLI095 and BAY11-7082 (p < 0.05) and additionally by SP00125 for AQP7 (p < 0.05). SB203580 had no effect on LPS-induced AQP3, AQP7 and AQP11 mRNA levels modulations. In conclusion, our results clearly show that many AQPs are expressed in murine 3T3-L1 adipocytes. Moreover, in DCs, LPS led to decreased AQP7 and AQP11 mRNA levels but to increased AQP3 mRNA levels, resulting from the Toll-like receptor 4 (TLR4)-induced activation of JNK and/or NFκB pathway.

Osmotic stress decreases aquaporin-4 expression in the human retinal pigment epithelial cell line, ARPE-19.

The regulation of water movement is of utmost importance for normal retinal function. Under physiological conditions, water is transported, dependent on the osmotic gradient, through the retinal pigment epithelial cell layer from the subretinal space to the choroid. The osmotic gradient has been found to be modified in eye diseases, thus leading to water accumulation in the subretinal space and the sensory retina, and subsequently contributing to the formation of macular oedema. Understanding the regulation of aquaporin expression is therefore crucial. In this study, we investigated the effects of hyperosmolarity on aquaporin-4 (AQP4) protein expression in the human retinal pigment epithelial cell line, ARPE­19. AQP4 expression was examined by PCR, western blot analysis and immunofluorescence. Ubiquitinylation was examined by immunoprecipitation. The results revealed that hyperosmotic stress rapidly decreased AQP4 expression in the ARPE-19 cells. The effect remained unmodified by lysosomal or mitogen-activated protein kinase inhibitors, but was reversed by proteasome inhibitors. However, no ubiquitinylation of AQP4 was detected. Our results suggest that hyperosmotic stress markedly reduces AQP4 expression possibly through a proteasome ubiquitinylation-independent pathway. This may represent an adaptation to hyperosmotic stress. The results presented in this study contribute to our understanding of the formation of macular oedema.

Pituitary adenylate cyclase activating peptide (PACAP) participates in adipogenesis by activating ERK signaling pathway.

Pituitary adenylate cyclase activating peptide (PACAP) belongs to the secretin/glucagon/vasoactive intestinal peptide (VIP) family. Its action can be mediated by three different receptor subtypes: PAC1, which has exclusive affinity for PACAP, and VPAC1 and VPAC2 which have equal affinity for PACAP and VIP. We showed that all three receptors are expressed in 3T3-L1 cells throughout their differentiation into adipocytes. We established the activity of these receptors by cAMP accumulation upon induction by PACAP. Together with insulin and dexamethasone, PACAP induced adipogenesis in 3T3-L1 cell line. PACAP increased cAMP production within 15 min upon stimulation and targeted the expression and phosphorylation of MAPK (ERK1/2), strengthened by the ERK1/2 phosphorylation being partially or completely abolished by different combinations of PACAP receptors antagonists. We therefore speculate that ERK1/2 activation is crucial for the activation of CCAAT/enhancer- binding protein ß (C/EBPß).

Murine 3T3-L1 adipocyte cell differentiation model: validated reference genes for qPCR gene expression analysis.

BACKGROUND: Analysis of gene expression at the mRNA level, using real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR), mandatorily requires reference genes (RGs) as internal controls. However, increasing evidences have shown that RG expression may vary considerably under experimental conditions. We sought for an appropriate panel of RGs to be used in the 3T3-L1 cell line model during their terminal differentiation into adipocytes. To this end, the expression levels of a panel of seven widely used RG mRNAs were measured by qRT-PCR. The 7 RGs evaluated were ß-actin (ACTB), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), hypoxanthine phosphoribosyl-transferase I (HPRT), ATP synthase H+ transporting mitochondrial F1 complex beta subunit (ATP-5b), tyrosine 3-monooxygenase/tryptophan 5- monooxygenase activation protein, zeta polypeptide (Ywhaz), Non-POU-domain containing octamer binding protein (NoNo), and large ribosomal protein L13a (RPL). METHODOLOGY/PRINCIPAL FINDINGS: Using three Excel applications, GeNorm, NormFinder and BestKeeper, we observed that the number and the stability of potential RGs vary significantly during differentiation of 3T3-L1 cells into adipocytes. mRNA expression analyses using qRT-PCR revealed that during the entire differentiation program, only NoNo expression is relatively stable. Moreover, the RG sets that were acceptably stable were different depending on the phase of the overall differentiation process (i.e. mitotic clonal expansion versus the terminal differentiation phase). RPL, ACTB, and Ywhaz, are suitable for terminal differentiation, whereas ATP-5b and HPRT, are suitable during mitotic clonal expansion. CONCLUSION: Our results demonstrate that special attention must be given to the choice of suitable RGs during the various well defined phases of adipogenesis to ensure accurate data analysis and that the use of several RGs is absolutely required. Consequently, our data show for the first time, that during mitotic clonal expansion, the most suitable RGs are ATP-5b, NoNo and HPRT, while during terminal differentiation the most suitable RGs are, NoNo, RPL, ACTB and Ywhaz.

G1/S Cyclins interact with regulatory subunit of PKA via A-kinase anchoring protein, AKAP95.

The mechanisms by which cyclins promote mammalian cell cycle progression have been a topic of intense investigation over the last decade. We previously described an interaction between D-type cyclins and A-kinase anchoring protein, AKAP95. Here, we demonstrate that AKAP95 can also bind cyclin E1. Association between AKAP95 and cyclins is displaced by CDKs. We show that these G(1)/S cyclins can interact with RII subunit of PKAalpha through AKAP95. The presence of alternate complexes cyclin-CDK and cyclin D/E-AKAP95-PKA.RIIalpha suggest different roles of G(1)/S cyclins and a wider biological importance of these interactions in cells.