SV40-transformed Cells Research Articles

Human Corneal Organotypic Cultures

Purpose. In order to test irritancy levels of commercial products, the Draize animal test has been the universally accepted choice. However, since this test uses live animals, many researchers have been trying to develop an alternative organotypic culture. These organotypic cultures, however, utilize animal cells, and it is our belief that a model using human cells would be more predictive for determining human irritancy levels. Methods. Primary human corneal epithelial cells and fibroblasts were separately isolated from a limbal rim and grown in culture. SV40-transformed mouse corneal endothelial cells were seeded onto a membrane and grown to confluence. A homogenous fibroblast/collagen mixture was then added and allowed to gel. After about a week, the epithelial cells were seeded on top of the gel and the constructs were kept submerged in culture for 3-7 days. The constructs were then airlifted to allow for the stratification of the epithelial cells. Following this, the constructs were either fixed and processed for methacrylate sectioning to study morphology, or they were frozen and sectioned for indirect immunofluorescence. Indirect immunofluorescence was performed with ZO-1, a marker of tight junctions; keratins 3 and 12, markers for differentiation; and laminin, a marker of basement membrane components. Initially, a SV40-transformed human endothelial cell line was to be used; however, it did not grow well in this culture system. Results. In these experiments, we tested two methods of isolating epithelial cells. One method, the explant technique-a method we previously used to isolate rabbit epithelial cells-could not be maintained beyond two passages, and when added to the construct only stratified to 2-3 layers of flattened cells. However, with the second method, the dispase technique, the epithelial cells obtained grew rapidly and could be maintained beyond two passages. These cells, when added to the construct, stratified to 5-7 layers and exhibited a more epithelium-like morphology. Staining with ZO-1 indicated that tight junctions in the superficial cells were formed. Staining with keratins 3 and 12 indicated that the epithelial cells were differentiating, and staining with laminin indicated that basement membrane components were being synthesized. Conclusion. Constructing an organotypic culture with human corneal cells is possible, and the morphology of the construct appears to be equivalent to a construct already created with bovine cells that has been used for irritancy studies.

Annexin A2 is a novel RNA-binding protein.

Annexin A2 (ANXA2) is a Ca(2+)-binding protein that is up-regulated in virally transformed cell lines and in human tumors. Here, we show that ANXA2 binds directly to both ribonucleotide homopolymers and human c-myc RNA. ANXA2 was shown to bind specifically to poly(G) with high affinity (K(d) = 60 nM) and not to poly(A), poly(C), or poly(U). The binding of ANXA2 to poly(G) required Ca(2+) (A(50%) = 10 microM). The presence of RNA in the immunoprecipitates of ANXA2 isolated from HeLa cells established that ANXA2 formed a ribonucleoprotein complex in vivo. Sucrose gradient analysis showed that ANXA2 associates with ribonucleoprotein complexes and not with polyribosomes. Reverse transcriptase-PCR identified c-myc mRNA as a component of the ribonucleoprotein complex formed by ANXA2 in vivo, and binding studies confirmed a direct interaction between ANXA2 and c-myc mRNA. Transfection of LNCaP cells with the ANXA2 gene resulted in the up-regulation of c-Myc protein. These findings identify ANXA2 as a Ca(2+)-dependent RNA-binding protein that interacts with the mRNA of the nuclear oncogene, c-myc.

Effects of RGD peptides on cells derived from the human eye

Purpose: To investigate the effects of Arg-Gly-Asp (RGD) peptides on cells derived from the human eye. Methods: SV40-transformed human corneal epithelial cells (SCE), SV40-transformed human lens epithelial cells (SLE), normal human keratocytes (NK), and human retinal pigment epithelial cells stably expressing human telomerase reverse transcriptase (TRPE) were used. A human corneal cDNA library was screened to isolate the human homologue of p130 Crk-associated substrate (Cas). Next, after these cells were attached to the substratum, RGD-containing soluble tetrapeptides Arg-Gly-Asp-Ser (RGDS) were added to the culture medium and morphological changes were observed. Results: A clone, H-2, consisting of 3228 nucleotides, with a long open reading frame (870 amino acid residues) was isolated, and determined to be the human homologue of Cas. After addition of the RGDS peptides, both SLE and TRPE detached from the plastic culture plate, but SCE and NK did not. Detached SLE and TRPE showed decreased levels of tyrosine phosphorylation in Cas. Conclusions: These results indicated that the signaling pathway through Cas played an important role in epithelial cell adhesion in the eye.

Aralin, a new cytotoxic protein from Aralia elata, inducing apoptosis in human cancer cells.

In this study, we purified a novel cytotoxic protein, aralin, from the shoots of Aralia elata. Aralin is composed of two subunits, A and B chains whose molecular weights are 29,100 and 32,200, respectively. In the assay using a normal human lung fibroblast cells (WI-38) and its SV40-transformed cells (VA-13), aralin demonstrated selective cytotoxicity against the virus-transformed cell line; the IC50 values of WI-38 and VA-13 were 10 and 0.8 ng/ml, respectively. Aralin showed positive response to DNA fragmentation in human lymphocyte HL-60 cells, and caspase specific inhibitors suppressed aralin-induced DNA fragmentation. These results indicate that the cytotoxicity of aralin is brought about primarily through the induction of apoptosis. Aralin also exhibited potent cytotoxic activity against various types of human cancer cell lines; cervical carcinoma cells (HeLa) proved the most sensitive, with an IC50 value of 0.08 ng/ml.

Evidence of expression of endotoxin receptors CD14, toll-like receptors TLR4 and TLR2 and associated molecule MD-2 and of sensitivity to endotoxin (LPS) in islet beta cells.

CD14, a GPI-linked membrane protein, is a component of the lipopolysaccharide (LPS) receptor complex, one of the pattern-recognizing receptors (PRR) expressed by myeloid lineage cells. Here we report that CD14, the functionally linked toll-like receptor molecules, TLR2 and TLR4, and the associated molecule MD-2 are expressed in endocrine cells of the human pancreatic islets. CD14 expression in human pancreatic islets was determined by immunofluorescence staining of tissue sections and primary cultures, and confirmed by flow cytometry of dispersed normal islets and SV40-transformed islet cells (HP62). The latter cells synthesized and secreted CD14 in response to lipopolysaccharide (LPS) in a time- and dose-dependent manner. Reverse transcription polymerase chain reaction (RT-PCR)-Southern was positive for CD14, TLR2, TLR4 and MD-2 in human pancreas, purified islets and HP62 cells. In vitro experiments using rat islets (also positive for CD14 by RT-PCR) and HP62 cells showed that LPS regulates glucose-dependent insulin secretion and induces inflammatory cytokines [interleukin (IL)-1alpha, IL-6 and tumour necrosis factor (TNF)-alpha]. The functional expression of CD14 and associated molecules in islet beta cells adds a new pathway that islet cells may follow to adjust their function to endotoxaemia situations and become vulnerable to the inflammatory events that occur during diabetogenic insulitis.

TRAIL-R2 (DR5) mediates apoptosis of synovial fibroblasts in rheumatoid arthritis.

TRAIL has been proposed as an anti-inflammatory cytokine in animal models of rheumatoid arthritis (RA). Using two agonistic mAbs specific for TRAIL-R1 (DR4) and TRAIL-R2 (DR5), we examined the expression and function of these death receptors in RA synovial fibroblast cells. The synovial tissues and primary synovial fibroblast cells isolated from patients with RA, but not those isolated from patients with osteoarthritis, selectively expressed high levels of cell surface DR5 and were highly susceptible to anti-DR5 Ab (TRA-8)-mediated apoptosis. In contrast, RA synoviocytes did not show increased expression of TRAIL-R1 (DR4), nor was there any difference in expression of Fas between RA and osteoarthritis synovial cells. In vitro TRA-8 induced apoptosis of RA synovial cells and inhibited production of matrix metalloproteinases induced by pro-inflammatory cytokines. In vivo TRA-8 effectively inhibited hypercellularity of a SV40-transformed RA synovial cell line and completely prevented bone erosion and cartilage destruction induced by these cells. These results indicate that increased DR5 expression and susceptibility to DR5-mediated apoptosis are characteristic of the proliferating synovial cells in RA. As highly proliferative transformed-appearing RA synovial cells play a crucial role in bone erosion and cartilage destruction in RA, the specific targeting of DR5 on RA synovial cells with an agonistic anti-DR5 Ab may be a potential therapy for RA.

Identification of androgen-regulated genes in SV40-transformed human hair dermal papilla cells

Background: Androgens are major regulators of human hair growth, which exert their effect on follicular epithelium via the mesenchyme-derived dermal papilla (DP) cells. However, very few data are available with regard to the genes regulated by androgen in DP cells. Objective: To investigate the differentially expressed genes by androgen in DP cells. Methods: Human hair DP cells were cultured and transformed with SV40 T antigen. Using cDNA representational difference analysis, androgen-regulated genes in SV40-transformed dermal papilla (SDP) cells were identified. Results: SDP cells revealed extended lifespan as compared with primary cultured DP cells. SDP cells expressed androgen receptor (AR) and showed androgen responsiveness. cDNA representational difference analysis followed by Northern blot analysis showed that heat shock cognate protein, Hsc70 was differentially expressed by dihydrotestosterone (DHT) treatment in SV40-transformed DP cells. Conclusion: These results suggest that Hsc70 may be involved in androgen action on DP cells.

A Question of Transformation: The Synovial Fibroblast in Rheumatoid Arthritis

Rheumatoid arthritis (RA) is a chronic, systemic, autoimmune disease with an unknown etiology. Both genetic and environmental factors contribute to disease susceptibility and the prevalence of RA is greater in women than in men. RA is characterized by initial vasculitis of the joint, followed by edema, infiltration of immune and inflammatory cells into the synovium, hyperplasia of the synovial lining, formation of lymph follicles, and development of the pannus. There is progressive cartilage destruction and finally erosion of the underlying bone. Current evidence suggests that fibroblast-like synoviocytes are one of the principle cells involved in pannus formation and cartilage degradation. Fibroblast-like synoviocytes in the rheumatoid synovium have characteristics similar to transformed cells and have been shown to proliferate in an anchorage-independent manner, lack contact inhibition, and express oncogenes and cell cycle proteins indicative of transformation. In vivo models have demonstrated that fibroblast-like synoviocytes can autonomously mediate cartilage destruction. In the article by Seemayer and colleagues in this issue of The American Journal of Pathology a direct comparison is made between SV40-transformed rheumatoid fibroblast-like synoviocytes and untransformed cells. 1 These experiments challenge the paradigm that rheumatoid synoviocytes are characterized by both aggressive growth and invasiveness. Of interest, the results demonstrate that fibroblast-like synoviocytes, capable of forming the pannus and invading cartilage, have an activated phenotype and secrete high levels of matrix-degrading enzymes, but have a limited capacity to proliferate. Conversely, SV40-transformed cells rapidly proliferate, secrete lower levels of matrix-degrading enzymes, display different levels of adhesion molecules, and demonstrate limited cartilage invasion. Thus these studies reveal an important dissociation between synoviocyte proliferation and invasiveness. This article reviews the tumor-like characteristics of fibroblast-like synoviocytes and discusses the contribution of synoviocytes to the pathogenesis of RA.

Heterogeneity of microvascular endothelial cells isolated from human term placenta and macrovascular umbilical vein endothelial cells

The present study compares some phenotypic and physiologic characteristics of microvascular and macrovascular endothelial cells from within one human organ. To this end microvascular endothelial cells from human full-term placenta (PLEC) were isolated using a new method and compared with macrovascular human umbilical vein endothelial cells (HUVEC) and an SV40-transformed placental venous endothelial cell line (HPEC-A2). PLEC were isolated by enzymatic perfusion of small placental vessels, purified on a density gradient and cultured subsequently. Histological sections of the enzyme-treated vessels showed a selective removal of the endothelial lining in the perfused placental cotyledons. The endothelial identity of the cells was confirmed by staining with the endothelial markers anti-von Willebrand factor, Ulex europaeus lectin and anti-QBEND10. The cells internalized acetylated low-density lipoprotein and did not show immunoreactivity with markers for macrophages, smooth muscle cells and fibroblasts. The spindle-shaped PLEC grew in swirling patterns similar to that described for venous placental endothelial cells. However, scanning electron microscopic examination clearly showed that PLEC remained elongated at the confluent state, in contrast to the more polygonal phenotype of HPEC-A2 and HUVEC that were studied in parallel. The amount of vasoactive substances (endothelin-1,2, thromboxane, angiotensin II, prostacyclin) released into the culture medium and the proliferative response to cytokines was more similar to human dermal microvessels (MIEC) derived from non-fetal tissue than to HUVEC. Potent mitogens such as vascular endothelial growth factors (VEGF121, VEGF165) and basic fibroblast growth factor (FGF-2) induced proliferation of all endothelial cell types. Placental growth factors PIGF-1 and PIGF-2 effectively stimulated cell proliferation on PLEC (142 +/- 7% and 173 +/- 10%) and MIEC (160 +/- 20% and 143 +/- 28%) in contrast to HUVEC (9 +/- 8% and 15 +/- 20%) and HPEC-A2 (15 +/- 7% and 24 +/- 6%) after 48 h incubation time under serum-free conditions. These data support evidence for (1) the microvascular identity of the isolated PLEC described in this study, and (2) the phenotypic and physiologic heterogeneity of micro- and macrovascular endothelial cells within one human organ.

Enhanced expression of Mcm proteins in cancer cells derived from uterine cervix.

Minichromosome maintenance proteins (Mcm) 2-7 play essential roles in eukaryotic DNA replication. Several reports have indicated the usefulness of Mcm proteins as markers of cancer cells in histopathological diagnosis. However, their mode of expression and pathophysiological significance in cancer cells remain to be clarified. We compared the level of expression of Mcm proteins among human HeLa uterine cervical carcinoma cells, SV40-transformed human fibroblast GM00637 cells and normal human fibroblast WI-38 cells. All the proteins examined were detected in HeLa and GM cells at 6-10 times the level found in WI-38 cells on average. This increase was observed both in total cellular proteins and in the chromatin-bound fraction. Consistently, Mcm2 mRNA was enriched in HeLa cells to approximately four times the level in WI-38 cells, and the synthesis of Mcm4, 6 and 7 proteins was accelerated in HeLa cells. Immunohistochemical studies of surgical materials from human uterine cervix showed that Mcm3 and 4 are ubiquitously expressed in cancer cells. Further, the positive rate and level of Mcm3 and 4 expression appeared to be higher in cancer cells than in normal proliferating cells of the uterine cervix and dysplastic cells, suggesting that they can be useful markers to distinguish these cells.

Notch-1 induction, a novel activity of SV40 required for growth of SV40-transformed human mesothelial cells.

We show that SV40 infection of human mesothelial cells directly causes overexpression of Notch-1, a key cell regulatory gene. Notch-1 induction is achieved at the transcriptional level and requires both the SV40 large T-antigen and the small t-antigen. Notch-1 upregulation is maintained in SV40-transformed human mesothelial clones and in SV40-positive mesotheliomas and derived cell lines. Activation of Notch-1 promotes cell cycle progression and it is required for the growth of SV40-transformed mesothelial cells. Our finding is relevant to the process of SV40-mediated human cell transformation, an effect that cannot be accounted for solely by SV40-Tag inhibition of Rb and p53.

Separation of two Cl– Currents in Cultured Human and Murine Mesangial Cells: Biophysical and Pharmacological Characteristics of ICl.vol and ICl.Ca

Mesangial cells, a combined smooth muscle- and fibroblast-like phenotype, are important regulators of renal function. These cells exist in a region of variable osmolarity and may require Cl^– channels for volume regulation. Additionally, Ca²⁺-activated Cl^– channels in these cells may participate in Ca²⁺-dependent contractile responses to vasoactive agonists. Relatively little, however, is known about mesangial cell Cl^– currents (I_Cl); including the biophysical description and pharmacological characterization. We used whole-cell patch clamp to study I_Cl in cultured human and SV40-transformed murine mesangial cells. I_Cl was measured in cells dialyzed and bathed with symmetrical N-methyl-D-glucamine chloride solutions to minimize cation currents. Dialysis with buffers to control intracellular Ca²⁺ ([Ca²⁺]_i), extracellular solutions of varied osmolarity, and manipulation of the transmembrane Cl^– gradient were used to separate two currents: I_Cl.vol (volume-sensitive), and I_Cl.Ca (Ca²⁺-activated). In symmetrical Cl^– with low [Ca²⁺]_i, I_Cl.vol was outwardly rectifying and modulated by osmolarity. I_Cl.vol demonstrated slight time- and voltage-dependent inactivation. In symmetrical Cl^– with elevated [Ca²⁺]_i and hypertonic bath, I_Cl.Ca was linear, but in asymmetrical Cl^– (low [Cl^–]_i) was outwardly rectifying and demonstrated time- and voltage-dependent activation. Permeability sequences for both I_Cl.vol and I_Cl.Ca were I^– > Br^– > Cl^– > F^–; however, there were differences in the relative magnitudes. Tamoxifen inhibited I_Cl.vol more potently than I_Cl.Ca, whereas niflumic acid inhibited I_Cl.Ca more potently than I_Cl.vol. We have separated and characterized two types of I_Cl in cultured human and murine mesangial cells. I_Cl.Ca and I_Cl.vol have different biophysical and pharmacological characteristics. These observations on I_Cl.Ca and I_Cl.vol may provide insight into mesangial cell reactivity and volume regulation.

The hemostatic agent ethamsylate enhances P-selectin membrane expression in human platelets and cultured endothelial cells

Ethamsylate possesses antihemorrhagic properties, but whether or not it directly activates blood platelets is unclear. Here we investigated the platelet activation potential of ethamsylate, by measuring membrane P-selectin expression with flow cytometry in human whole blood and also by immunofluorescence imaging of isolated human platelets. Moreover, we measured membrane P-selectin expression in the SV40-transformed aortic rat endothelial cell line (SVAREC) and 14C-ethamsylate membrane binding and/or uptake in platelets and endothelial cells. Whole blood flow cytometry showed a modest, but statistically significant increase by ethamsylate in the percentage of platelets expressing P-selectin (from 2% to 4–5%, p<0.05). Immunofluorescence showed a sizable (39%) and significant ( p<0.01) enhancement of P-selectin expression at the lowest concentration of ethamsylate tested (1 μM), with maximal enhancement of P-selectin expression (75–90%) at 10 μM ethamsylate. Similar results were obtained in SVAREC endothelial cells. 14C-ethamsylate specifically bound to platelets and endothelial cell membranes, without significant uptake into the cell interior. In conclusion, ethamsylate enhances membrane P-selectin expression in human platelets and in cultured endothelial cells. Ethamsylate specifically binds to some protein receptor in platelet and endothelial cell membranes, receptor which can signal for membrane P-selectin expression. These results support the view that ethamsylate acts on the first step of hemostasis, by improving platelet adhesiveness and restoring capillary resistance.

A highly potent 26,27-Hexafluoro-1a,25-dihydroxyvitamin D3 on calcification in SV40-transformed human fetal osteoblastic cells.

26,27-hexafluoro-1a,25-dihydroxyvitamin D3 (F6-D3) has been reported to be 5-10 times more potent than 1a,25-dihydroxyvitamin D3[1,25(OH)2D3] in biological systems in vivo and in vitro. However, the effect of F6-D3 on bone formation has yet to be clarified. In the present study, we investigated the effect of F6-D3 on SV40-transfected human fetal osteoblastic cells (SV-HFO) and found it to be about 100 times greater than that of 1,25(OH)2D3 in stimulating calcification. F6-D3 was also about 100 times more effective than 1,25(OH)2D3 in enhancing the expression of mRNA for alkaline phosphatase (ALP), osteocalcin (OCN), and osteopontin (OPN). In the presence of 10?8 M F6-D3 and 10?6 M 1,25(OH)2D3, the calcification began on day 9 and increased up to day 19. Expression of mRNA for ALP and OCN reached a maximum on day 4 and thereafter declined. On the other hand, when osteoblastic cells were incubated with a low level of [1b-3H]-F6-D3- or [1b-3H]-1,25(OH)2D3, each radioactive peak could not be detected. However, on the incubation of osteoblastic cells and radioactive substrate in the presence of ketoconazole, a selective inhibitor of CYP24, a clear peak for each substrate was detected. This suggested that F6-D3 as well as 1,25(OH)2D3 is metabolized by CYP24. Osteoblastic cells were incubated with 10?8 M[1b-3H]-F6-D3 or 10?8 M[1b-3H]-1,25(OH)2D3 for 4, 9, and 14 days. A small peak of 1,25(OH)2D3 was observed and thereafter its level decreased. In addition, two unknown peaks increased when the culture period was extended. In the case of F6-D3, peaks of F6-D3 and 26,27-hexafluoro-23-oxo-1a,25(OH)2D3(23-oxo-F6) were clearly detected, the latter being about 4 times higher than the former. Both peaks was retained up to day 14. The amount of unlabeled F6-D3 and 23-oxo-F6 calculated from the specific radioactivity in the cells may be similar to the amount of 1,25(OH)2D3 and its metabolites. The strong activity of F6-D3 in stimulating calcification may be due to the fact that F6-D3 is much more potent than 1,25(OH)2D3 in enhancing the expression of mRNA for ALP, OCN, and OPN and that the amount of F6-D3 and 23-oxo-F6 accumulated in the cells is much greater than that of 1,25(OH)2D3 and its metabolite.

Activation of ATM and phosphorylation of p53 by heat shock.

p53 protein is phosphorylated in response to various stresses. Here we examined phosphorylation of p53 protein in normal human diploid cells after heat shock at 43 degrees C for 2 h. We found that heat shock stimulates phosphorylation of p53 at Ser15 but not at Ser20, while X-irradiation at 4 Gy and 10 J/m(2) of UV induces phosphorylation of p53 at Ser15 and less significantly at Ser20. Increased phosphorylation of Ser15 was also observed in heat shocked GM638, the SV40-transformed human fibroblast cell line. Although X-ray irradiation induced phosphorylation of Ser6, 9, 20, and 37 in GM638 cells, heat shock did not affect the phosphorylation level of these serines. We observed little or no phosphorylation of p53 at Ser15 in two primary ataxia telangiectasia fibroblast cells, that are defective in ATM. Using an in vitro kinase assay, we confirmed that immunoprecipitated ATM from both heat-shocked and X-irradiated normal human diploid cells can phosphorylate p53 at Ser15 to a similar extent. These results indicate that heat shock induces phosphorylation of p53, especially at Ser15, and its phosphorylation is mediated by ATM kinase.

Termination of lifespan of SV40-transformed human fibroblasts in crisis is due to apoptosis.

Normal human fibroblasts in culture have a limited lifespan, ending in replicative senescence. Introduction of SV40 sequences encoding large T antigen and small t antigen into pre-senescent cells results in an extension of lifespan for an additional 20-30 population doublings. Rare clones of SV40-transformed cells are capable of indefinite growth and are described as immortal; however, the great majority of SV40-transformed cells terminate this extended lifespan in cell death, termed "crisis." We have examined the properties of cells in crisis to obtain further insights into mechanism of cell death and immortalization. Populations at the terminal cell passage show a balance between cell replication and cell death over a period of several weeks, with a progressive increase in cells undergoing cell death. During this period, there is less than a 3-fold increase in attached cell number, with two stages being identifiable on the basis of the focal pattern of cell survival. We also demonstrate that cells in crisis are undergoing apoptosis based on TUNEL assay, subG1 DNA content, annexin V reactivity, and activation of caspases 3 and 8. We suggest a model whereby SV40-transformed cells acquire increased sensitivity to apoptosis based on changes in properties which activate caspase 8 in addition to changes previously described involving shortening of telomeric sequences. While only telomere stabilization could be clearly shown to be essential for survival of cells through crisis, the extended period of cell replication and altered gene expression observed in SV40-transformed cells during crisis are compatible with other genetic alterations in immortal cells.

DDB Accumulates at DNA Damage Sites Immediately after UV Irradiation and Directly Stimulates Nucleotide Excision Repair

Damaged DNA-binding protein, DDB, is a heterodimer of p127 and p48 with a high specificity for binding to several types of DNA damage. Mutations in the p48 gene that cause the loss of DDB activity were found in a subset of xeroderma pigmentosum complementation group E (XP-E) patients and have linked to the deficiency in global genomic repair of cyclobutane pyrimidine dimers (CPDs) in these cells. Here we show that with a highly defined system of purified repair factors, DDB can greatly stimulate the excision reaction reconstituted with XPA, RPA, XPC.HR23B, TFIIH, XPF.ERCC1 and XPG, up to 17-fold for CPDs and approximately 2-fold for (6-4) photoproducts (6-4PPs), indicating that no additional factor is required for the stimulation by DDB. Transfection of the p48 cDNA into an SV40-transformed human cell line, WI38VA13, was found to enhance DDB activity and the in vivo removal of CPDs and 6-4PPs. Furthermore, the combined technique of recently developed micropore UV irradiation and immunostaining revealed that p48 (probably in the form of DDB heterodimer) accumulates at locally damaged DNA sites immediately after UV irradiation, and this accumulation is also observed in XP-A and XP-C cells expressing exogenous p48. These results suggest that DDB can rapidly translocate to the damaged DNA sites independent of functional XPA and XPC proteins and directly enhance the excision reaction by core repair factors.

Molecular Basis for the Dual Mitochondrial and Cytosolic Localization of Alanine:Glyoxylate Aminotransferase in Amphibian Liver Cells

To gain further insights into the molecular basis of the evolution of alanine:glyoxylate aminotransferase (AGT) intracellular targeting in vertebrates, we have studied the molecular basis of its dual mitochondrial and cytosolic distribution in amphibian liver cells. The AGT gene in Xenopus laevis encodes a polypeptide of 415 amino acids, which includes a 24-residue N-terminal mitochondrial targeting sequence (MTS), at either end of which are located two in-frame potential translation start sites. This MTS is necessary to target Xenopus AGT and sufficient to target a green fluorescent fusion protein to mitochondria in transfected COS cells. The C-terminal tripeptide (KKM), despite being similar to the nonconsensus type 1 peroxisomal targeting sequence in human AGT (KKL), was unable to target Xenopus AGT or human AGT to peroxisomes. The Xenopus AGT gene produces two types of transcript. The longer form encodes a polypeptide that contains the MTS and is targeted to mitochondria. The shorter form encodes a polypeptide that does not contain the MTS and remains in the cytosol. These results are discussed not only in terms of the molecular evolution of AGT targeting but also in terms of the ancillary requirements for the peroxisomal targeting of human AGT.

Ras and Rac as activators of reactive oxygen species (ROS).

The investigation into the relationship between reactive oxygen species (ROS) and the signaling pathways of the Ras family of small GTPases has experienced a recent surge in interest. Known to cause DNA damage, and implicated in disease processes as wide-ranging as atherosclerosis and kidney disease, ROS have appeared in unlikely capacities within the Ras signaling pathway. Eschewing their evil ways, ROS have been shown to be essential for mitogenesis in Ras-transformed cells, and have been identified as mediators of a Rac-dependent anti-apoptotic signal in cells microinjected with activated Ras (,). The beneficence of ROS on Ras-transformed cells carries with it the macroscopic potential for selection and growth promotion of cancerous cells. Several investigations have suggested such a role for ROS, specifically superoxide (O2 s), in a Ras-independent manner. Populations of SV40-transformed cells have been observed to undergo phenotypic reversion when transfected with MnSOD,an enzyme that converts superoxide to hydrogen peroxide. Moreover, several investigations have demonstrated decreased levels of MnSOD in populations of cancerous cells (,). Together, these studies suggest a requirement for superoxide in the maintenance of the transformed state.

Identification of human multidrug resistance protein 1 (MRP1) mutations and characterization of a G671V substitution.

The multidrug resistance protein 1 (MRP1) belonging to the ATP-binding cassette (ABC) superfamily of transport proteins can confer resistance to multiple natural product drugs and methotrexate in human tumor cells. In addition, MRP1 is expressed in normal tissues acting as an efflux pump for glutathione, glucuronate, and sulfate conjugates and may thus influence the pharmacokinetic properties of many drugs. Using polymerase chain reaction-single-strand conformation polymorphism analysis, we screened 36 Caucasian volunteers for mutations in the coding exons of the MRP1 gene, including the adjacent intron sequences. Among several mutations found, two are expected to cause amino acid substitutions. One of these mutations (G671V) was of special interest because it is located near the first nucleotide binding domain. To determine whether this mutation caused a change in the MRP1 phenotype, a mutant MRP1 expression vector was constructed and transfected into SV40-transformed human embryonic kidney cells (HEKSV293T) and the transport properties of the mutant protein were examined. Transport of the MRP1 substrates leukotriene C4, 17beta-estradiol 17beta-(D)-glucuronide, and estrone sulfate by membrane vesicles prepared from transiently transfected HEKSV293T cells was comparable to that of wild-type MRP1.