A novel C-terminal motif is necessary for the export of the vasopressin V1b/V3 receptor to the plasma membrane

Little is known about endoplasmic reticulum (ER) export signals, particularly those of members of the G-protein-coupled receptor family. We investigated the structural motifs involved in membrane export of the human pituitary vasopressin V1b/V3 receptor. A series of V3 receptors carrying deletions and point mutations were expressed in AtT20 corticotroph cells. We analyzed the export of these receptors by monitoring radioligand binding and by analysis of a V3 receptor tagged with both green fluorescent protein and Myc epitopes by a novel flow cytometry-based method. This novel method allowed us to quantify total and membrane-bound receptor expression. Receptors lacking the C terminus were not expressed at the cell surface, suggesting the presence of an export motif in this domain. The distal C terminus contains two di-acidic (DXE) ER export motifs; however, mutating both these motifs had no effect on the V3 receptor export. The proximal C terminus contains a di-leucine (345)LL(346) motif surrounded by the hydrophobic residues Phe(341), Asn(342), and Leu(350). The mutation of one or more of these five residues abolished up to 100% of the receptor export. In addition, these mutants colocalized with calnexin, demonstrating that they were retained in the ER. Finally, this motif was sufficient to confer export properties on a CD8alpha glycoprotein-V3 receptor chimera. In conclusion, we have identified a novel export motif, FN(X)(2)LL(X)(3)L, in the C terminus of the V3 receptor.     

Expression and function of Toll-like receptor on T cells

Toll is the founder of a group of pattern recognition receptors, which play a critical role in the innate immunity in Drosophila. At least 13 distinct Toll-like receptors (TLRs), recognising pathogen-associated molecular pattern (PAMPs), have now been identified in humans. Most investigations on TLRs have focused on cells of the innate system. We report here that na?ve human T cells expressed high levels of cell surface TLR2 after activation by anti-T cell receptor (TCR) antibody and interferon-alpha. Activated cells produced elevated levels of cytokines in response to the TLR2 ligand, bacterial lipopeptide (BLP). Furthermore, CD4(+)CD45RO(+) memory T cells from peripheral blood constitutively expressed TLR2 and produced IFNgamma in response to BLP. BLP also markedly enhanced the proliferation and IFNgamma production by CD45RO(+) T cells in the presence of IL-2 or IL-15. Thus, TLR2 serves as a co-stimulatory receptor for antigen-specific T cell development and participates in the maintenance of T cell memory. This suggests that pathogens, via their PAMPs, may contribute directly to the perpetuation and activation of long term T cell memory in both antigen dependent and independent manner.     

Mapping of an inducible element in the T cell receptor V beta 2 promoter.

The murine V beta 2 promoter was analyzed for an element regulating phorbol ester inducibility of the TCR beta chain gene. In transient expression analysis of 5' nested deleted fragments of the V beta 2 promoter, the TPA-inducible element mapped between -85 and -42. The -85 to -62 oligo conferred 12-0-tetradecanoylphorbol-13-acetate (TPA) inducibility to the heterologous TPA-uninducible thymidine kinase promoter. The -85 to -62 region contained an AP-1 site (-85 to -72) and inverted repeat motif (-72 to -62). The AP-1 site required the 3' flanking inverted repeat region for conferring optimal inducibility. In vitro transcribed and translated jun/fos heterodimers bind to the V beta 2 AP-1 motif with a 16-fold lower affinity as compared to the collagenase AP-1 motif. This explains the inability of the V beta 2 AP-1 motif to confer optimal TPA inducibility by itself. The affinity of jun/fos heterodimers for the V beta 2 AP-1 motif was not increased by the presence in cis of the inverted repeat motif. The 3' flanking inverted repeat binds the ets transactivator but not jun/fos heterodimers. The demonstrated cooperativity between the AP-1 and the 3' flanking sequence to confer TPA inducibility can thus be explained by the individual contributions of jun/fos and ets transactivators.     

A versatile interaction platform on the Mex67-Mtr2 receptor creates an overlap between mRNA and ribosome export

The transport receptor Mex67-Mtr2 functions in mRNA export, and also by a loop-confined surface on the heterodimer binds to and exports pre-60S particles. We show that Mex67-Mtr2 through the same surface that recruits pre-60S particles interacts with the Nup84 complex, a structural module of the nuclear pore complex devoid of Phe-Gly domains. In vitro, pre-60S particles and the Nup84 complex compete for an overlapping binding site on the loop-extended Mex67-Mtr2 surface. Chemical crosslinking identified Nup85 as the subunit in the Nup84 complex that directly binds to the Mex67 loop. Genetic studies revealed that this interaction is crucial for mRNA export. Notably, pre-60S subunit export impaired by mutating Mtr2 or the 60S adaptor Nmd3 could be partially restored by second-site mutation in Nup85 that caused dissociation of Mex67-Mtr2 from the Nup84 complex. Thus, the Mex67-Mtr2 export receptor employs a versatile binding platform on its surface that could create a crosstalk between mRNA and ribosome export pathways.     

Impact of a first-order riparian zone on nitrogen removal and export from an agricultural ecosystem

Riparian zones are reputed to be effective at preventing export of agricultural groundwater nitrogen (N) from local ecosystems. This is one impetus behind riparian zone regulations and initiatives. However, riparian zone function can vary under different conditions, with varying impacts on the regional (and ultimately global) environment. Rates of groundwater delivery to the surface appear to have significant effects on the N-removing capabilities of a riparian zone. Research conducted at a first-order agricultural watershed with a well-defined riparian zone in the Maryland coastal plain indicates that more than 2.5 kg/day of nitrate-N can be exported under moderate-to-high stream baseflow conditions. The total nitrate-N load that exits the system increases with increasing flow not simply because of the greater volume of water export. Stream water nitrate-N concentrations also increase by more than an order of magnitude as flow increases, at least during baseflow. This appears to be largely the result of changes in dominant groundwater delivery mechanisms. Higher rates of groundwater exfiltration lessen the contact time between nitrate-carrying groundwater and potentially reducing riparian soils. Subsurface preferential flow paths, in the wetland and adjacent field, also strongly influence N removal. Simple assumptions regarding riparian zone function may be inadequate because of complexities observed in response to changing hydrologic conditions.     

A model of the ACE2 structure and function as a SARS-CoV receptor

The angiotensin-converting enzyme 2 (ACE2) is an important regulator of the renin-angiotensin system and was very recently identified as a functional receptor for the SARS virus. The ACE2 sequence is similar (sequence identities 43% and 35%, and similarities 61% and 55%, respectively) to those of the testis-specific form of ACE (tACE) and the Drosophila homolog of ACE (AnCE). The high level of sequence similarity allowed us to build a robust homology model of the ACE2 structure with a root-mean-square deviation from the aligned crystal structures of tACE and AnCE less than 0.5A. A prominent feature of the model is a deep channel on the top of the molecule that contains the catalytic site. Negatively charged ridges surrounding the channel may provide a possible binding site for the positively charged receptor-binding domain (RBD) of the S-glycoprotein, which we recently identified [Biochem. Biophys. Res. Commun. 312 (2003) 1159]. Several distinct patches of hydrophobic residues at the ACE2 surface were noted at close proximity to the charged ridges that could contribute to binding. These results suggest a possible binding region for the SARS-CoV S-glycoprotein on ACE2 and could help in the design of experiments to further elucidate the structure and function of ACE2.     

Cbl promotes ubiquitination of the T cell receptor zeta through an adaptor function of Zap-70

Triggering of the T cell antigen receptor (TCR).CD3 complex induces its ubiquitination. However, the molecular events that lead to ubiquitin conjugation to these cell surface molecules have not been defined. Here we report that Cbl, a RING-type E3 ubiquitin-protein ligase, promotes ubiquitination of TCR zeta chain, which requires its functional variant Src homology 2 domain and an intact RING finger. The tyrosine kinase Zap-70, which binds to both TCR zeta and Cbl, plays an adaptor role in these events. Mutations in TCR zeta, Zap-70, or Cbl that disrupt the interaction between TCR zeta and Zap-70 or between Zap-70 and Cbl reduce ubiquitination of TCR zeta. Our results suggest a novel mechanism by which Cbl negatively regulates T cell development and activation by inducing ubiquitination of the TCR.CD3 components.     

Purification of the human apical conjugate export pump MRP2 reconstitution and functional characterization as substrate-stimulated ATPase.

The multidrug resistance protein MRP2 (ABCC2) acts as an ATP-dependent conjugate export pump in apical membranes of polarized cells and confers multidrug resistance. Purified MRP2 is essential for the detailed functional characterization of this member of the family of ATP-binding cassette (ABC) transporter proteins. In human embryonic kidney cells (HEK293), we have permanently expressed MRP2 containing an additional C-terminal (His)6-tag. Immunoblot and immunofluorescence analyses detected the MRP2-(His)6 overexpressing clones. Isolated membrane vesicles from the MRP2-(His)6-expressing cells were active in ATP-dependent transport of the glutathione S-conjugate leukotriene C4 and were photoaffinity-labelled with 8-azido-[alpha-32P]ATP. MRP2-(His)6 was solubilized from membranes of MRP2-(His)6-cells and purified to homogeneity in a three-step procedure using immobilized metal affinity chromatography, desalting, and immunoaffinity chromatography. The identity of the pure MRP2-(His)6 was verified by MS analysis of tryptic peptides. The purified MRP2-(His)6 glycoprotein was reconstituted into proteoliposomes and showed functional activity as ATPase in a protein-dependent manner with a Km for ATP of 2.1 mM and a Vmax of 25 nmol ADP x mg MRP2-1 x min-1. This ATPase activity was substrate-stimulated by oxidized and reduced glutathione and by S-decyl-glutathione. Future studies using pure MRP2 reconstituted in proteoliposomes should allow further insight into the molecular parameters contributing to MRP2 transport function and to define its intracellular partners for transport and multidrug resistance.     

Impact of iron particles in groundwater on the UV inactivation of bacteriophages MS2 and T4

AIMS: To investigate the impact of iron particles in groundwater on the inactivation of two model viruses, bacteriophages MS2 and T4, by 254-nm ultraviolet (UV) light. METHODS AND RESULTS: One-litre samples of groundwater with high iron content (from the Indianapolis Water Company, mean dissolved iron concentration 1.3 mg l(-1)) were stirred vigorously while exposed to air, which oxidized and precipitated the dissolved iron. In parallel samples, ethylenediaminetetra-acetic acid (EDTA) was added to chelate the iron and prevent formation of iron precipitate. The average turbidity in the samples without EDTA (called the 'raw' samples) after 210 min of stirring was 2.7 +/- 0.1 NTU while the average turbidity of the samples containing EDTA (called the 'preserved' samples) was 1.0 +/- 0.1 NTU. 'Raw' and 'preserved' samples containing bacteriophage MS2 were exposed to 254-nm UV light at doses of 20, 40, or 60 mJ (cm(2))(-1), while samples containing bacteriophage T4 were exposed to 2 or 5 mJ (cm(2))(-1), using a low pressure UV collimated beam. The UV inactivation of both phages in the 'raw' groundwater was lower than in the EDTA-'preserved' groundwater to a statistically significant degree (alpha = 0.05), due to the association of phage with the UV-absorbing iron precipitate particles. A phage elution technique confirmed that a large fraction of the phage that survived the UV exposures were particle-associated. CONCLUSIONS: Phages that are associated with iron oxide particles in groundwater are shielded from UV light to a measurable and statistically significant degree at a turbidity level of 2.7 NTU when the phage particle association is induced under experimental conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: While the particle association of the phage in this study was induced experimentally, the findings provide further evidence that certain particles in natural waters and wastewaters (e.g. iron oxide particles) may have the potential to shield viruses from UV light.     

A predominance of the T cell receptor V gamma 2/V delta 2 subset in human mycobacteria-responsive T cells suggests germline gene encoded recognition.

Little is known about the nature of Ag recognition by the TCR-gamma delta. The recent observation that gamma delta T cells preferentially recognize mycobacterial Ag provides a model to examine the molecular basis of gamma delta-TCR recognition. Here, examination of the Mycobacteria-stimulated peripheral blood T cells with TCR-specific mAb revealed a predominance of T cells bearing V gamma 2/V delta 2 gene products. PCR cloning and sequence analysis of the TCR chains demonstrated extensive junctional diversity indicating that the response was polyclonal. The marked in vitro gamma delta T cell response to Mycobacteria was also detected in newborns before encounters with foreign Ag and exclusively involved the same V-gene usage observed in adults. Together, these results suggest that a major mechanism of gamma delta T cell reactivity involves recognition mediated by germline-encoded segments of the TCR.     

Asialo GM1 as an accessory molecule determining the function and reactivity of cytotoxic T lymphocytes

The expression and function of asialo-GM1 (AsGM1) in alloreactive cytotoxic T lymphocytes (CTL) was studied. We have shown previously that the cytotoxic reactions mediated by AsGM1+-cloned CTL were blocked by anti-AsGM1 or by purified AsGM1. To further determine the role of AsGM1 in CTL-mediated cytotoxicity, we examined the correlation between this blocking effect and the expression of AsGM1 on effector and target cells. Now we found that the blocking by anti-AsGM1 was largely dependent on the expression of AsGM1 on the effector cells in a dose-dependent fashion. The expression of AsGM1 on target cells had only little effect on the blocking of cytotoxic reactions by anti-AsGM1 or AsGM1. A threefold difference was seen in the blocking of AsGM1+ and AsGM1- targets. The observation was in sharp contrast to the effectors as no blocking was ever seen with AsGM1- CTL. Similar to CTL effectors, we found that the expression of AsGM1 and L3T4 were mutually excluded on mitogen-activated T cells, despite the fact that they could coexpress in resting T cells. The expression of AsGM1 on CTL effectors was associated with the antigen-nonspecific natural killer (NK)-like or lymphokine-activated killer (LAK)-like activity exerted by the alloreactive CTL. All AsGM1+ CTL possessed LAK activity against antigen-unrelated tumor targets, and the AsGM1- CTL only displayed antigen-specific alloreactivity. The LAK activity was associated with the expression of AsGM1 on effectors, and was not related to the AsGM1 expression on target cells. These findings indicate that the AsGM1 expressed on alloreactive CTL may function as an accessory molecule for T-cell receptors in the antigen-specific alloreactive cytotoxicity mediated by AsGM1+ CTL. The expression of AsGM1 may also be related to the activation of an NK-like apparatus in these CTL. Therefore, AsGM1 not only may be involved in cytotoxic reactions mediated by AsGM1+ CTL, it may also modulate the specificity of the CTL cytotoxicity.     

CD69 expression as an index of T-cell function: assay standardization, validation and use in monitoring immune recovery

BACKGROUND: CD69 is a surrogate marker of T-cell responsiveness to mitogen and Ag stimulus and can be used as a measure of T-lymphocyte activation. Quantitative flow cytometric determination of CD69 expression on T lymphocytes has several advantages over traditional lymphocyte proliferation assays, but this method has not yet been standardized for clinical applications. METHODS: We qualified a commercially available assay using the manufacturer's procedures for measurement of T-cell response to a mitogen (PHA), superantigen (Staphylococcus endotoxin B; SEB) and Ca(2+) ionophore (phorbyl myristate acetate; PMA) with peripheral blood from healthy volunteers. Following this, we tested the usefulness of the assay in determining T-cell responses to PHA and SEB for six immunocompromised patients. RESULTS: Healthy volunteers showed 17-fold increases in T-cell CD69 Ab bound per cell (ABC) with PHA stimulation compared with the baseline. SEB was also an effective T-cell activating agent, increasing CD69 ABC by 5-fold, comparable with results obtained with PMA stimulation. PHA- and SEB-stimulated T-cell CD69 ABC for patients 100 days post-BM transplant were generally below 1 SD of that from healthy volunteers. SEB-stimulated T-cell CD69 expression was significantly depressed for CD8(+) T cells while CD4(+) T-cell responses to SEB were generally within 1 SD of the mean for healthy volunteers. DISCUSSION: These results suggest that quantitative measurement of CD69 surface expression by flow cytometry is a useful diagnostic tool for detailed assessment of T-lymphocyte and subset activation.     

Serine mutations in transmembrane V of the dopamine D1 receptor affect ligand interactions and receptor activation.

Several serines present in transmembrane domain V are conserved among members of the G-protein-coupled receptor family that bind catecholamines. Two of these serines that are present in the beta-adrenergic receptor were previously shown by site-directed mutagenesis to affect agonist binding and receptor activation (Strader, C. D., Candelore, M. R., Hill, W. S., Sigal, I. S., and Dixon, R. A. F. (1989) J. Biol. Chem. 264, 13572-13578). We investigated the role of the serines present in transmembrane V of another catecholamine receptor, the dopamine D1 receptor, by site-directed mutagenesis, and the results show that mutations at serines 198, 199, and 202 affect dopamine binding. The substitution of serine 198 or serine 199 by an alanine also affects the binding of several other agonist and antagonist dopaminergic compounds while an alanine substitution at serine 202 has no effect on the binding of these compounds. Moreover, each single serine mutation decreased the maximal cAMP accumulation elicited by a dopamine D1 partial agonist. These results suggest that serines present in transmembrane V of the D1 receptor affect ligand interactions and receptor signal transduction, but not entirely in the manner that would be predicted from the model proposed for the beta-adrenergic receptor.     

Characterization of the betaherpesviral pUL69 protein family reveals binding of the cellular mRNA export factor UAP56 as a prerequisite for stimulation of nuclear mRNA export and for efficient viral replication

UL69 of human cytomegalovirus (HCMV) encodes a pleiotropic transactivator protein and has a counterpart in every member of the Herpesviridae family thus far sequenced. However, little is known about the conservation of the functions of the nuclear phosphoprotein pUL69 in the homologous proteins of other betaherpesviruses. Therefore, eukaryotic expression vectors were constructed for pC69 of chimpanzee cytomegalovirus, pRh69 of rhesus cytomegalovirus, pM69 of murine cytomegalovirus, pU42 of human herpesvirus 6, and pU42 of elephant endotheliotropic herpesvirus. Indirect immunofluorescence experiments showed that all pUL69 homologs expressed by these vectors were localized to the cell nucleus. Coimmunoprecipitation experiments identified homodimerization as a conserved feature of all homologs, whereas heterodimerization with pUL69 was restricted to its closer relatives. Further analyses demonstrated that pC69 and pRh69 were the only two homologs that functioned, like pUL69, as viral-mRNA export factors. As we had reported recently that nucleocytoplasmic shuttling and interaction with the cellular DExD/H-box helicases UAP56 and URH49 were prerequisites for the nuclear-mRNA export activity of pUL69, the homologs were characterized with regard to these properties. Heterokaryon assays demonstrated nucleocytoplasmic shuttling for all homologs, and coimmunoprecipitation and mRNA export assays revealed that the interaction of UAP56 and/or URH49 with pC69 or pRh69 was required for mRNA export activity. Moreover, characterization of HCMV recombinants harboring mutations within the N-terminal sequence of pUL69 revealed a strong replication defect of viruses expressing pUL69 variants that were deficient in UAP56 binding. In summary, homodimerization and nucleocytoplasmic shuttling activity were identified as conserved features of betaherpesviral pUL69 homologs. UAP56 binding was shown to represent a unique characteristic of members of the genus Cytomegalovirus that is required for efficient replication of HCMV.     

Impact of the Vitamin D3 receptor on growth-regulatory pathways in mammary gland and breast cancer

1,25-Dihydroxyvitamin D₃ (1,25(OH)₂D₃) interacts with the Vitamin D₃ receptor (VDR) to modulate proliferation and apoptosis in a variety of cell types, including breast cancer cells. In this review, we discuss three issues related to the role of the VDR in growth control: first, whether mammary glands lacking VDR exhibit abnormal growth; second, whether the VDR is essential for induction of apoptosis by 1,25(OH)₂D₃; and third, whether VDR up-regulation can sensitize cells to 1,25(OH)₂D₃. Studies from our laboratory have demonstrated that mammary glands from VDR knockout (VDR KO) mice exhibit accelerated growth and branching during puberty, pregnancy and lactation as compared to wild-type (WT) mice. In addition, involution after weaning, a process driven by epithelial cell apoptosis, proceeds at a slower rate in VDR KO mice compared to WT mice. Using cells isolated from VDR KO and WT mice, we report that both normal and transformed mammary cells derived from WT mice are growth inhibited by 1,25(OH)₂D₃, however, cells derived from VDR KO mice are completely unresponsive to 1,25(OH)₂D₃. In human breast cancer cells, we have identified a variety of agents, including steroid hormones, phytoestrogens and growth factors, that up-regulate VDR expression and enhance sensitivity to 1,25(OH)₂D₃-mediated growth inhibition. Collectively, these studies support a role for 1,25(OH)₂D₃ and the VDR in negative growth regulation of both normal mammary gland and breast cancer cells.     

Assessing the role of the T cell receptor beta gene enhancer in regulating coding joint formation during V(D)J recombination

To assess the role of the T cell receptor (TCR) beta gene enhancer (Ebeta) in regulating the processing of VDJ recombinase-generated coding ends, we assayed TCRbeta rearrangement of Ebeta-deleted (DeltaEbeta) thymocytes in which cell death is inhibited via expression of a Bcl-2 transgene. Compared with DeltaEbeta, DeltaEbeta Bcl-2 thymocytes show a small accumulation of TCRbeta standard recombination products, including coding ends, that involves the proximal Dbeta-Jbeta and Vbeta14 loci but not the distal 5' Vbeta genes. These effects are detectable in double negative pro-T cells, predominate in double positive pre-T cells, and correlate with regional changes in chromosomal structure during double negative-to-double positive differentiation. We propose that Ebeta, by driving long range nucleoprotein interactions and the control of locus expression and chromatin structure, indirectly contributes to the stabilization of coding ends within the recombination processing complexes. The results also illustrate Ebeta-dependent and -independent changes in chromosomal structure, suggesting distinct modes of regulation of TCRbeta allelic exclusion depending on the position within the locus.     

Differences in transferrin receptor function between normal developing and transformed myogenic cells as revealed by differential effects of phorbol ester on receptor distribution and rates of iron uptake

The effects of the tumor promotor, 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA), on the intra- and extracellular distribution of transferrin receptors and rates of iron uptake were studied in normal developing myogenic cells and myogenic cells transformed with a temperature-sensitive strain of the Rous sarcoma virus. In normal developing cells PMA was found to increase the rate of iron uptake by 15-30%. There was, however, no effect on transferrin receptor distribution, suggesting that the increase in iron uptake was due to stimulation of the rate of receptor cycling. In contrast, in transformed myogenic cells, PMA had no effect even at concentrations 10 times those effective in normal myogenic cells. The specificity of PMA was demonstrated by comparison with 4 alpha-phorbol which had no effect compared with the control cells which were incubated with dimethyl sulfoxide, the solvent used to dissolve the phorbols. These results indicate a functional difference in the transferrin receptor between normal and transformed myogenic cells. The data for normal myogenic cells are similar to those previously reported for normal erythroid cells, but differ from those for some transformed cell lines in which phorbol esters were shown to cause internalization of transferrin receptors.     

Impact of subdomain D1 of the short form S1b of the human prolactin receptor on its inhibitory action on the function of the long form of the receptor induced by prolactin

Background

Long-form (LF) homodimers of the human prolactin receptor (PRLR) mediate prolactin's diverse actions. Short form S1b inhibits the LF function through heterodimerization. Reduced S1b/LF-ratio in breast cancer could contribute to tumor development/progression. Current work defines the structural and functional relevance of the D1 domain of S1b on its inhibitory function on prolactin-induced LF function.

Methods

Studies were conducted using mutagenesis, promoter/signaling analyses, bioluminescence resonance energy transfer (BRET) and molecular modeling approaches.

Results

Mutation of E69 in D1 S1b or adjacent residues at the receptor surface near to the binding pocket (S) causes loss of its inhibitory effect while mutations away from this region (A) or in the D2 domain display inhibitory action as the wild-type. All S1b mutants preserved prolactin-induced Jak2 activation. BRET reveals an increased affinity in D1 mutated S1b (S) homodimers in transfected cells stably expressing LF. In contrast, affinity in S1b homodimers with either D1 (A) or D2 mutations remained unchanged. This favors LF mediated signaling induced by prolactin. Molecular dynamics simulations show that mutations (S) elicit major conformational changes that propagate downward to the D1/D2 interface and change their relative orientation in the dimers.

Conclusions

These findings demonstrate the essential role of D1 on the S1b structure and its inhibitory action on prolactin-induced LF-mediated function.

General significance

Major changes in receptor conformation and dimerization affinity are triggered by single mutations in critical regions of D1. Our structure–function/simulation studies provide a basis for modeling and design of small molecules to enhance inhibition of LF activation for potential use in breast cancer treatment.     

Impact of proliferative activity and tumorigenic conversion on mitochondrial function of fibroblasts in 2D and 3D culture

The purpose of the present study was to examine mitochondrial function in differently transformed cells relative to their tumorigenic state and proliferative activity in vitro. An established two-step carcinogenesis model consisting of immortal and tumorigenic rat embryo fibroblasts that can be cultured as monolayers and multicellular spheroids was investigated. Flow cytometric measurements were carried out using the two mitochondrial-specific fluorochromes rhodamine 123 (Rh123) and 10-N-nonyl acridine orange (NAO), in combination with the DNA dye Hoechst 33342 for simultaneous cell cycle analysis. Since the accumulation of Rh123 depends on mitochondrial membrane potential, Rh123 fluorescence intensity gives an estimate of mitochondrial activity per cell, as determined by both overall mitochondrial function and mass. In contrast, NAO uptake reflects mitochondrial mass only, as it binds to cardiolipin in the inner mitochondrial membrane independently of membrane potential. Aliquots of cell suspensions derived from exponential monolayer, confluent monolayer, and a range of sizes of multicellular spheroids were stained with either Rh123 or NAO and Hoechst 33342, then mitochondrial mass and activity per unit cell volume and cellular DNA content were measured by flow cytometry. Differences in the average mitochondrial activity per cell in different cell lines and culture conditions were primarily due to alterations in cell volume. Importantly, tumorigenic conversion by ras-transfection did not consistently change mitochondrial activity per unit cell volume. The mitochondrial mass per unit cell volume increased for all cells when cellular quiescence was induced, either in monolayers or spheroids. However, mitochondrial function (activity/mass) decreased when cells became quiescent, resulting in a positive correlation between mitochondrial function and S-phase fraction, independent of transformation status or culture condition. We conclude that mitochondrial function reflects proliferative activity rather than tumorigenic conversion.