In-depth immune profiling of peripheral blood mononuclear cells in patients with pancreatic ductal adenocarcinoma reveals discriminative immune subpopulations.

Pancreatic ductal adenocarcinoma (PDAC) has a dismal prognosis with a 5-year survival of less than 10%. More knowledge of the immune response developed in patients with PDAC is pivotal to develop better combination immune therapies to improve clinical outcome. In this study, we used mass cytometry time-of-flight to undertake an in-depth characterization of PBMCs from patients with PDAC and examine the differences with healthy controls and patients with benign diseases of the biliary system or pancreas. Peripheral blood mononuclear cells from patients with PDAC or benign disease are characterized by the increase of pro-inflammatory cells, as CD86+ classical monocytes and memory T cells expressing CCR6+ and CXCR3+, associated with T helper 1 (Th1) and Th17 immune responses, respectively. However, PBMCs from patients with PDAC present also an increase of CD39+ regulatory T cells and CCR4+CCR6-CXCR3- memory T cells, suggesting Th2 and regulatory responses. Concluding, our results show PDAC develops a multifaceted immunity, where a proinflammatory component is accompanied by regulatory responses, which could inhibit potential antitumor mechanisms.

Prognostic and predictive value of human equilibrative nucleoside transporter 1 (hENT1) in extrahepatic cholangiocarcinoma: a translational study.

Introduction: Effective (neo) adjuvant chemotherapy for cholangiocarcinoma is lacking due to chemoresistance and the absence of predictive biomarkers. Human equilibrative nucleoside transporter 1 (hENT1) has been described as a potential prognostic and predictive biomarker. In this study, the potential of rabbit-derived (SP120) and murine-derived (10D7G2) antibodies to detect hENT1 expression was compared in tissue samples of patients with extrahepatic cholangiocarcinoma (ECC), and the predictive value of hENT1 was investigated in three ECC cell lines. Methods: Tissues of 71 chemonaïve patients with histological confirmation of ECC were selected and stained with SP120 or 10D7G2 to assess the inter-observer variability for both antibodies and the correlation with overall survival. Concomitantly, gemcitabine sensitivity after hENT1 knockdown was assessed in the ECC cell lines EGI-1, TFK-1, and SK-ChA-1 using sulforhodamine B assays. Results: Scoring immunohistochemistry for hENT1 expression with the use of SP120 antibody resulted in the highest interobserver agreement but did not show a prognostic role of hENT1. However, 10D7G2 showed a prognostic role for hENT1, and a potential predictive role for gemcitabine sensitivity in hENT1 in SK-ChA-1 and TFK-1 cells was found. Discussion: These findings prompt further studies for both preclinical validation of the role of hENT1 and histochemical standardization in cholangiocarcinoma patients treated with gemcitabine-based chemotherapy.

Spatial immune composition of tumor microenvironment in patients with pancreatic cancer.

This study examined the composition of the immune microenvironment at different sites within resected pancreas specimens from patients with pancreatic ductal adenocarcinoma (PDAC). Therefore, single-cell suspensions were made from fresh tumor and non-tumorous tissue. Fourteen patients were included from whom twelve PDAC and five non-tumorous samples were obtained. These samples were analyzed with a nineteen marker panel on the Aurora spectral flow cytometer. Furthermore, slides from formalin-fixed paraffine PDACs of eight additional patients were stained with eight markers and analyzed by multispectral imaging. These corresponded to central tumor, periphery of the tumor, i.e., invasive front and resected lymph node and were divided into tumor and adjacent tissue. In the single-cell suspension, a decreased ratio between lymphoid and myeloid cells and between M1 and M2 macrophages was observed in the tumor tissue compared to non-tumorous tissue. Furthermore, an increase in CD169 + macrophages in patients undergoing neoadjuvant therapy was found. Using immunofluorescence, more macrophages compared to T cells were observed, as well as a lower ratio of CD8 to M2 macrophage, a higher ratio of CD4-CD8 T cells and a higher ratio of immune-suppressive cells to pro-inflammatory cells in the PDAC area compared to the adjacent non-tumorous tissue. Finally, there were more immune-suppressive cells in the central tumor area compared to the invasive front. In conclusion, we show a gradient in the immune-suppressive environment in PDAC from most suppressive in the central tumor to least suppressive in distant non-tumorous tissue.

Prediction Model for Early-Stage Pancreatic Cancer Using Routinely Measured Blood Biomarkers.

Importance: Accurate risk prediction models using routinely measured biomarkers-eg, carbohydrate antigen 19-9 (CA19-9) and bilirubin serum levels-for pancreatic cancer could facilitate early detection of pancreatic cancer and prevent potentially unnecessary diagnostic tests for patients at low risk. An externally validated model using CA19-9 and bilirubin serum levels in a larger cohort of patients with pancreatic cancer or benign periampullary diseases is needed. Objective: To assess the discrimination, calibration, and clinical utility of a prediction model using readily available blood biomarkers (carbohydrate antigen 19-9 [CA19-9] and bilirubin) to distinguish early-stage pancreatic cancer from benign periampullary diseases. Design, Setting, and Participants: This diagnostic study used data from 4 academic hospitals in Italy, the Netherlands, and the UK on adult patients with pancreatic cancer or benign periampullary disease treated from 2014 to 2022. Analyses were conducted from September 2022 to February 2023. Exposures: Serum levels of CA19-9 and bilirubin from samples collected at diagnosis and before start of any medical intervention. Main Outcomes and Measures: Discrimination (measured by the area under the curve [AUC]), calibration, and clinical utility of the prediction model and the biomarkers, separately. Results: The study sample comprised 249 patients in the development cohort (mean [SD] age at diagnosis, 67 [11] years; 112 [45%] female individuals), and 296 patients in the validation cohort (mean [SD] age at diagnosis, 68 [12] years; 157 [53%] female individuals). At external validation, the prediction model showed an AUC of 0.89 (95% CI, 0.84-0.93) for early-stage pancreatic cancer vs benign periampullary diseases, and outperformed CA19-9 (difference in AUC [ΔAUC], 0.10; 95% CI, 0.06-0.14; P < .001) and bilirubin (∆AUC, 0.07; 95% CI, 0.02-0.12; P = .004). In the subset of patients without elevated tumor marker levels (CA19-9 <37 U/mL), the model showed an AUC of 0.84 (95% CI, 0.77-0.92). At a risk threshold of 30%, decision curve analysis indicated that performing biopsies based on the prediction model was equivalent to reducing the biopsy procedure rate by 6% (95% CI, 1%-11%), without missing early-stage pancreatic cancer in patients. Conclusions and Relevance: In this diagnostic study of patients with pancreatic cancer or benign periampullary diseases, an easily applicable risk score showed high accuracy for distinguishing early-stage pancreatic cancer from benign periampullary diseases. This model could be used to assess the added diagnostic and clinical value of novel biomarkers and prevent potentially unnecessary invasive diagnostic procedures for patients at low risk.

Diagnostic accuracy and added value of blood-based protein biomarkers for pancreatic cancer: A meta-analysis of aggregate and individual participant data.

Background: Novel blood-based protein biomarkers may be of value for efficient, accurate, and non-invasive diagnosis of pancreatic cancer. This study assesses the diagnostic accuracy of newly recognized blood-based protein biomarkers for detecting pancreatic cancer, and investigates their added value to CA19-9, the common blood-based biomarker in clinical use for pancreatic cancer. Methods: PubMed, Embase, Web of Science, and the Wiley/Cochrane Library were systematically searched from inception until June 2022. A meta-analysis of aggregate and individual participant data was conducted using frequentist and Bayesian hierarchical random-effects models. The added clinical utility of protein biomarkers was investigated using bootstrap bias-corrected decision curve analyses. Findings: Aggregate data from 28 primary studies (6127 participants) were included, of which 8 studies (1790 participants) provided individual participant data. CA19-9 was significantly more accurate than MIC-1 for distinguishing pancreatic cancer from benign disease (AUC, 0.83 vs 0.74; relative diagnostic odds ratio [rDOR], 2.10 [95% CI, 0.98-4.48]; p = 0.002), THBS2 (AUC, 0.87 vs 0.69; rDOR, 4.53 [2.16-9.39]; p < 0.0001), TIMP-1 (AUC, 0.91 vs 0.70; rDOR, 8.00 [3.81-16.9]; p < 0.0001), OPN (AUC, 0.89 vs 0.74; rDOR, 4.22 [1.13-15.6]; p < 0.0001), ICAM-1 (AUC, 0.91 vs 0.68; rDOR 9.30 [0.87-99.5]; p < 0.0001), and IGFBP2 (AUC, 0.91 vs 0.68; rDOR, 4.48 [0.78-24.3]; p < 0.0001). The addition of these novel protein biomarkers to CA19-9 did not significantly improve the AUC, and resulted in minor increases or limited decreases in clinical utility. Interpretation: Novel protein biomarkers have moderate diagnostic accuracy, do not outperform CA19-9 in differentiating pancreatic cancer from benign disease, and show limited added clinical value to CA19-9. We propose recommendations to aid the development of minimally invasive diagnostic tests with sufficient clinical utility to improve the management of patients with suspected pancreatic cancer. Funding: Bennink Foundation, Dutch Cancer Foundation (KWF Kankerbestrijding), and AIRC.

Factors that influence the pancreatic and duodenal microbiome in patients undergoing pancreatic surgery.

BACKGROUND/OBJECTIVES: This study examined the correlation between pancreatic microbiome and patients characteristics. Furthermore, we compared different duodenal materials to examine their reflection of the pancreatic microbiome. METHODS: Patients undergoing pancreatic surgery were included in the study. Characteristics of those patients were prospectively registered and sterile pancreatic biopsies were collected during surgery. After completion of the resection, duodenal fluid, -tissue and -swab were collected. Bacterial DNA was extracted and analyzed with IS-pro assay. RESULTS: Paired samples of 51 patients were available for evaluation, including pancreatic biopsies from all patients, 22 duodenal fluids, 21 duodenal swabs and 11 duodenal tissues. The pancreatic microbiome consisted mostly of Proteobacteria followed by Firmicutes, Actinobacteria, Fusobacteria and Verrucomicrobia (FAFV) and Bacteroidetes. On species level, Enterococcus faecalis, Escherichia coli, and Enterobacter-Klebsiella were most abundant. In pancreatic biopsies, the total bacterial load and Proteobacteria load were significantly higher in patients with biliary drainage (54618.0 vs 5623.5; 9119.0 vs 2067.1). Patients who used proton pump inhibitors had a significantly higher total bacterial load (115964.7 vs 8495.8), more FAFV (66862.9 vs 1890.1), more Proteobacteria (24245.9 vs 2951.4) and more Bacteroidetes (542.5 vs 25.8). The head of the pancreas contained significantly more bacteria (21193.4 vs 2096.8) and more FAFV (5225.7 vs 19.0) compared to the tail, regardless of biliary drainage. Furthermore, the microbiome of all duodenal materials showed a weak correlation with the pancreatic microbiome. CONCLUSION: Biliary drainage, use of proton pump inhibitors, and anatomic location of the pancreatic biopsy influence the pancreatic microbiome. Furthermore, the duodenal microbiome does not suffice as a surrogate for the pancreatic microbiome.

The Diagnostic Value of the CA19-9 and Bilirubin Ratio in Patients with Pancreatic Cancer, Distal Bile Duct Cancer and Benign Periampullary Diseases, a Novel Approach.

Distinction of pancreatic ductal adenocarcinoma (PDAC) in the head of the pancreas, distal cholangiocarcinoma (dCCA), and benign periampullary conditions, is complex as they often share similar clinical symptoms. However, these diseases require specific management strategies, urging improvement of non-invasive tools for accurate diagnosis. Recent evidence has shown that the ratio between CA19-9 and bilirubin levels supports diagnostic distinction of benign or malignant hepatopancreaticobiliary diseases. Here, we investigate the diagnostic value of this ratio in PDAC, dCCA and benign diseases of the periampullary region in a novel fashion. To address this aim, we enrolled 265 patients with hepatopancreaticobiliary diseases and constructed four logistic regression models on a subset of patients (n = 232) based on CA19-9, bilirubin and the ratio of both values: CA19-9/(bilirubin-1). Non-linearity was investigated using restricted cubic splines and a final model, the 'Model Ratio', based on these three variables was fitted using multivariable fractional polynomials. The performance of this model was consistently superior in terms of discrimination and calibration compared to models based on CA19-9 combined with bilirubin and CA19-9 or bilirubin alone. The 'Model Ratio' accurately distinguished between malignant and benign disease (AUC [95% CI], 0.91 [0.86-0.95]), PDAC and benign disease (AUC 0.91 [0.87-0.96]) and PDAC and dCCA (AUC 0.83 [0.74-0.92]) which was confirmed by internal validation using 1000 bootstrap replicates. These findings provide a foundation to improve minimally-invasive diagnostic procedures, ultimately ameliorating effective therapy for PDAC and dCCA.

Heterogeneity and plasticity of cancer-associated fibroblasts in the pancreatic tumor microenvironment.

Pancreatic ductal adenocarcinoma (PDAC) is an aggressive disease with a notably poor prognosis, in urgent need of improved treatment strategies. The desmoplastic PDAC tumor microenvironment (TME), marked by a high concentration of cancer-associated-fibroblasts (CAFs), is a dynamic part of PDAC pathophysiology which occasions a variety of effects throughout the course of pancreatic tumorigenesis and disease evolution. A better understanding of the desmoplastic TME and CAF biology in particular, should provide new opportunities for improving therapeutics. That CAFs have a tumor-supportive role in oncogenesis is well known, yet research evidence has shown that CAFs also have tumor-repressive functions. In this review, we seek to clarify the intriguing heterogeneity and plasticity of CAFs and their ambivalent role in PDAC tumorigenesis and progression. Additionally, we provide recommendations to advance the implementation of CAF-directed PDAC care. An improved understanding of CAFs' origins, spatial location, functional diversity, and marker determination, as well as CAF behavior during the course of PDAC progression and metastasis will provide essential knowledge for the future improvement of therapeutic strategies for patients suffering from PDAC.

NUC-1031 in biliary tract cancer: from bench to bedside and back?

In preclinical models of biliary tract cancer, NUC-1031 showed less potency than gemcitabine, no correlation with potential biomarkers and only moderate additive interaction in combination with cisplatin. These findings should prompt further careful pharmacological and translational studies to better define the purported therapeutic advantage of NUC-1031 over gemcitabine. That would be a more cautious approach than the phase III clinical trial which is planning to enrol 828 patients with biliary tract tumours to compare gemcitabine/cisplatin "conventional" treatment with or without NUC-1031.

Combined Expression of Plasma Thrombospondin-2 and CA19-9 for Diagnosis of Pancreatic Cancer and Distal Cholangiocarcinoma: A Proteome Approach.

BACKGROUND: Minimally invasive diagnostic biomarkers for patients with pancreatic ductal adenocarcinoma (PDAC) and distal cholangiocarcinoma (dCCA) are warranted to facilitate accurate diagnosis. This study identified diagnostic plasma proteins based on proteomics of tumor secretome. MATERIALS AND METHODS: Secretome of tumor and normal tissue was collected after resection of PDAC and dCCA. Differentially expressed proteins were measured by mass spectrometry. Selected candidate biomarkers and carbohydrate antigen 19-9 (CA19-9) were validated by enzyme-linked immunosorbent assay in plasma from patients with PDAC (n = 82), dCCA (n = 29), benign disease (BD; n = 30), and healthy donors (HDs; n = 50). Areas under the curve (AUCs) of receiver operator characteristic curves were calculated to determine the discriminative power. RESULTS: In tumor secretome, 696 discriminatory proteins were identified, including 21 candidate biomarkers. Thrombospondin-2 (THBS2) emerged as promising biomarker. Abundance of THBS2 in plasma from patients with cancer was significantly higher compared to HDs (p < .001, AUC = 0.844). Combined expression of THBS2 and CA19-9 yielded the optimal discriminatory capacity (AUC = 0.952), similarly for early- and late-stage disease (AUC = 0.971 and AUC = 0.911). Remarkably, this combination demonstrated a power similar to CA19-9 to discriminate cancer from BD (AUC = 0.764), and THBS2 provided an additive value in patients with high expression levels of bilirubin. CONCLUSION: Our proteome approach identified a promising set of candidate biomarkers. The combined plasma expression of THBS2/CA19-9 is able to accurately distinguish patients with PDAC or dCCA from HD and BD. IMPLICATIONS FOR PRACTICE: The combined plasma expression of thrombospondin-2 and carbohydrate antigen 19-9 is able to accurately diagnose patients with pancreatic cancer and distal cholangiocarcinoma. This will facilitate minimally invasive diagnosis for these patients by distinguishing them from healthy individuals and benign diseases.

Proteomic Analysis of miR-195 and miR-497 Replacement Reveals Potential Candidates that Increase Sensitivity to Oxaliplatin in MSI/P53wt Colorectal Cancer Cells.

Most patients with advanced colorectal cancer (CRC) eventually develop resistance to systemic combination therapy. miR-195-5p and miR-497-5p are downregulated in CRC tissues and associated with drug resistance. Sensitization to 5-FU, oxaliplatin, and irinotecan by transfection with miR-195-5p and miR-497-5p mimics was studied using cell viability and clonogenic assays in cell lines HCT116, RKO, DLD-1, and SW480. In addition, proteomic analysis of transfected cells was implemented to identify potential targets. Significantly altered proteins were subjected to STRING (protein-protein interaction networks) database analysis to study the potential mechanisms of drug resistance. Cell viability analysis of transfected cells revealed increased sensitivity to oxaliplatin in microsatellite instable (MSI)/P53 wild-type HCT116 and RKO cells. HCT116 transfected cells formed significantly fewer colonies when treated with oxaliplatin. In sensitized cells, proteomic analysis showed 158 and 202 proteins with significantly altered expression after transfection with miR-195-5p and miR-497-5p mimics respectively, of which CHUK and LUZP1 proved to be coinciding downregulated proteins. Resistance mechanisms of these proteins may be associated with nuclear factor kappa-B signaling and G1 cell-cycle arrest. In conclusion, miR-195-5p and miR-497-5p replacement enhanced sensitivity to oxaliplatin in treatment naïve MSI/P53 wild-type CRC cells. Proteomic analysis revealed potential miRNA targets associated with the cell-cycle which possibly bare a relation with chemotherapy sensitivity.

Activation of LINE-1 Retrotransposon Increases the Risk of Epithelial-Mesenchymal Transition and Metastasis in Epithelial Cancer.

Epithelial cancers comprise 80-90% of human cancers. During the process of cancer progression, cells lose their epithelial characteristics and acquire stem-like mesenchymal features that are resistant to chemotherapy. This process, termed the epithelial-mesenchymal transition (EMT), plays a critical role in the development of metastases. Because of the unique migratory and invasive properties of cells undergoing the EMT, therapeutic control of the EMT offers great hope and new opportunities for treating cancer. In recent years, a plethora of genes and noncoding RNAs, including miRNAs, have been linked to the EMT and the acquisition of stem cell-like properties. Despite these advances, questions remain unanswered about the molecular processes underlying such a cellular transition. In this article, we discuss how expression of the normally repressed LINE-1 (or L1) retrotransposons activates the process of EMT and the development of metastases. L1 is rarely expressed in differentiated stem cells or adult somatic tissues. However, its expression is widespread in almost all epithelial cancers and in stem cells in their undifferentiated state, suggesting a link between L1 activity and the proliferative and metastatic behaviour of cancer cells. We present an overview of L1 activity in cancer cells including how genes involved in proliferation, invasive and metastasis are modulated by L1 expression. The role of L1 in the differential expression of the let-7 family of miRNAs (that regulate genes involved in the EMT and metastasis) is also discussed. We also summarize recent novel insights into the role of the L1-encoded reverse transcriptase enzyme in epithelial cell plasticity that suggest it might be a potential therapeutic target that could reverse the EMT and the metastasis-associated stem cell-like properties of cancer cells.

Enhancement of vertebrate cardiogenesis by a lectin from perivitelline fluid of horseshoe crab embryo.

Cardiac myocytes are the first cells to differentiate during the development of a vertebrate embryo. A wide variety of molecules take part in various steps in this process. While exploring biologically active molecules from marine sources, we found that a constituent of perivitelline fluid from embryos of the Indian horseshoe crab can enhance growth and differentiation of chick embryonic heart. We have purified the factor and identified the cardiac promoting molecule to be a novel lectin. We show that this molecule influences cardiac development by increasing the number of cells constituting the heart and by modulating the expression of several cardiac development regulatory genes in chick embryos. Using mouse embryonic stem cells we show that the cardiac myocyte-enhancing capacity of this molecule extends to mammals and its effects can be blocked using methylated sugars. This molecule may prove to be an important tool in the study of cardiomyocyte differentiation.

Nestin-specific green fluorescent protein expression in embryonic stem cell-derived neural precursor cells used for transplantation.

Expression of the enhanced green fluorescent protein (EGFP) under control of a thymidine kinase promoter/nestin second intron was specifically detected in nestin immunoreactive neural precursor cells after selection of murine embryonic stem (ES) cells in chemically defined medium. Allowing differentiation in vitro, the capacity of these cells to give rise to astroglia, oligodendroglia, and neurones was investigated. After intracerebral transplantation, long-lasting integration of precursor cells into the host tissue was observed, serving as a pool for successive neuronal and glial differentiation. EGFP expression by ES cell-derived neural precursor cells may be a valuable tool to optimize protocols for maintenance and expansion of these cells in vitro as well as in vivo after intracerebral transplantation. In addition, preparative fluorescence-activated cell sorting of EGFP-labeled neural precursor cells should be useful for standardization of a donor cell population for cell replacement therapies.

Protein and mRNA expression of serum and glucocorticoid-dependent kinase 1 in metanephrogenesis.

Abstract Expression of serum and glucocorticoid-dependent kinase 1 (SGK1) during development in mouse kidney (embryonic day [E] 14 to postnatal day [P] 1) was studied by in situ hybridization and immunofluorescence. In whole embryos, SGK1 mRNA was highly abundant in the developing metanephros, where SGK1 mRNA was expressed in the ureteric buds of the branching collecting duct system and in the mesenchymal blastema-derived comma- and s-shaped bodies. In E14 kidneys, SGK1 protein was below detection level, whereas at day E16, ureteric buds, s-shaped bodies and outgrowing loops of Henle expressed detectable amounts of SGK1 protein. SGK1 protein was also expressed in E16 primary tubules of the collecting duct system. In P1 kidneys, no or only faint SGK1 protein expression was apparent in comma- and s-shaped bodies, whereas SGK1 was continuously expressed by medullary collecting ducts. In conclusion, SGK1 is developmentally expressed in metanephrogenesis. High expression in developing collecting duct and in blastema-derived comma- and s-shaped bodies suggests a dual function of SGK1 in maturation of the reabsorbing collecting duct epithelium and in epithelial transition of the blastema cells.

The expression and regulation of depolarization-activated K+ channels in the insulin-secreting cell line INS-1.

The aim of the present study was to characterize depolarization-activated outward currents in insulin-secreting INS-1 cells and to investigate the role of K+ channels other than the KATP channels in the regulation of insulin release. Outward currents were inhibited by 4-aminopyridine (4-AP, 10 mmol/l), tetraethylammonium (TEA, 10 mmol/l) and tetrapentylammonium (TPeA, 100 mumol/l) by 55.1 +/- 3.8% (n = 3), 78.1 +/- 3.2% (n = 6) and 98.7 +/- 0.8% (n = 5), respectively. Margatoxin (5 nmol/l) and charybdotoxin (3 mumol/l) had no effect. 4-AP inhibited mainly a fast-activating, slowly inactivating current, whereas the TEA- and TPeA-sensitive current components were slowly activating and non-inactivating. Forskolin and the forskolin analogue 1,9-dideoxyforskolin, which does not stimulate adenylyl cyclase, also inhibited the outward current, suggesting a direct effect on the channels. Using reverse transcriptase polymerase chain reaction (RT/PCR). Kv channel mRNAs of Kv1.4, Kv1.5, Kv2.1, Kv2.2, Kv3.1 and Kv3.2 were detected whereas other Kv channels, Kv1.1, Kv1.2, Kv1.3, Kv1.6 and Kv3.4 were not detected. Insulin secretion in the presence of tolbutamide (100 mumol/l) was increased by 4-AP, TEA and TPeA by 65%, 41% and 150%, respectively. Basal secretion was not affected by these blockers. Our study reveals that the opening of voltage-dependent K+ channels negatively controls insulin secretion in depolarized cells, probably by shortening the action potential thus reducing Ca2+ influx.

Tissue variant effects of heme inhibitors on the mouse cytochrome c oxidase gene expression and catalytic activity of the enzyme complex.

The in vivo effects of heme biosynthesis inhibitors, succinylacetone and CoCl2 on the cytochrome c oxidase (COX) gene expression and enzyme activity in different mouse tissues were investigated. Succinylacetone and CoCl2 showed tissue-specific differences in their ability to modulate heme aa3 content. A single dose of succinylacetone treatment for 8 h reduced the heme aa3 content of kidney mitochondria with no effect on the liver. CoCl2 treatment for 8 h, however, selectively affected the heme aa3 level in the liver. Reduced mitochondrial heme aa3 with both treatments was accompanied by approximately 50% reduced, mitochondrial genome-encoded COX I and II mRNAs and nuclear genome-encoded COX Vb mRNAs, but no change in COX IV mRNA level. Use of isolated mouse liver and brain mitochondrial systems showed a 50-80% reduction in mitochondrial transcription and translation rates in heme-depleted tissues. Blue native gel electrophoresis followed by immunoblot analysis showed that the complex from heme-depleted tissues contained a 30-50% reduction in levels of subunits I, IV, Vb and near normal levels of subunit VIc, indicating altered subunit content. Treatment of submitochondrial particles with protein kinase A and ATP resulted in partial dissociation of COX, suggesting a mechanistic basis for the reduced subunit content of the complex from heme-depleted tissues. Surprisingly, the enzyme from heme-depleted tissues showed twofold to fourfold higher turnover rates for cytochrome c oxidation, suggesting alterations in the kinetic characteristics of the enzyme following heme reduction. This is probably the first evidence that the tissue heme level regulates not only the mammalian COX gene expression, but also the catalytic activity of the enzyme, probably by affecting its stability.