Overexpression of the RNA-binding proteins Lin28B and IGF2BP3 (IMP3) is associated with chemoresistance and poor disease outcome in ovarian cancer.

BACKGROUND: RNA-binding proteins have an important role in messenger RNA (mRNA) regulation during tumour development and carcinogenesis. In the present study, we examined the insulin-like growth factor 2 mRNA-binding proteins (IGF2BPs; hereafter refered to as IMPs) and Lin28 family expressions in epithelial ovarian carcinoma (EOC) patients and correlated their expression levels with the response to chemotherapy, hCTR1 expression and patient survival. METHODS: Patients clinical information, real-time RT-PCR, immunohistochemistry, western blot, Transwell migration invasion assays, and cytotoxicity assays were used. RESULTS: From 140 EOC patients, high expression of IMP3 or Lin28B was associated with poor survival, and women diagnosed at advanced stages with elevated IMP3 and Lin28B were at higher risk of developing chemoresistance. High IMP3 levels combined with high Lin28B levels significantly correlated with the poorest 5-year survival rates. Knockdown of IMP3 or Lin28B decreased cell proliferation, migration, and invasion, and increased the platinum sensitivity, but not taxol sensitivity, of ovarian cancer cells through increased expression of hCTR1, a copper transporter involved in platinum uptake. High expression of hCTR1 correlated with low expression of IMP3/Lin28B and better progression-free survival in advanced-stage EOC patients. CONCLUSION: Testing for a combination of elevated IMP3 and Lin28B levels could further facilitate the identification of a patient subgroup with the worst prognosis.

Clinical implications of insulin-like growth factor 1 system in early-stage cervical cancer.

This study was aimed to identify the expression and the correlation of insulin-like growth factor-1 (IGF-1) system and their prognostic impacts in cervical cancer. Seventy-two patients with early-stage cervical cancer were eligible. We obtained the serum levels of total IGF-1 and IGF binding protein-3 (IGFBP-3) by enzyme-linked immunosorbent assay and the expression of IGF-1 receptor (IGF-1R) in cancerous tissue by immuno-fluorescent (IF) stains. The 5-year recurrence-free and overall survival rates were significantly lower (P=0.003 and P=0.01, respectively) among patients with high-grade expression of tissue IGF-1R, compared with those with low-grade expression. After adjustment for other factors, preoperative serum total IGF-1 or IGFBP-3 levels failed to predict cancer death and recurrence. High-grade expression of IGF-1R and elevated preoperative squamous cell carcinoma antigen level were independent predictors of both death and recurrence, and combination of both factors could further help identify the subgroup of patients at higher death risk. The IF staining indicates the colocalisation of IGF-1 and IGF-1R in the cancerous tissues, whereas the IGF-1R expression is not correlated with circulating levels of IGF-1 or IGFBP-3. In early-stage cervical cancer, IGF-1 system may have a paracrine or autocrine function and the adverse impacts on prognosis by IGF-1R overexpression are implicated.

Nonconservative amino acid substitution variants exist at polymorphic frequency in DNA repair genes in healthy humans.

The removal or repair of DNA damage has a key role in protecting the genome of the cell from the insults of cancer-causing agents. This was originally demonstrated in individuals with the rare genetic disease xeroderma pigmentosum, the paradigm of cancer genes, and subsequently in the relationship between mismatch repair and colon cancer. Recent reports suggest that individuals with less dramatic reductions in the capacity to repair DNA damage are observed at polymorphic frequency in the population; these individuals have an increased susceptibility to breast, lung, and skin cancer. We report initial results from a study to estimate the extent of DNA sequence variation among individuals in genes encoding proteins of the DNA repair pathways. Nine different amino acid substitution variants have been identified in resequencing of the exons of three nucleotide excision repair genes (ERCC1, XPD, and XPF), a gene involved in double-strand break repair/recombination genes (XRCC3), and a gene functioning in base excision repair and the repair of radiation-induced damage (XRCCI). The frequencies for the nine different variant alleles range from 0.04 to 0.45 in a group of 12 healthy individuals; the average allele frequency is 0.17. The potential that this variation, and especially the six nonconservative amino acid substitutions occurring at residues that are identical in human and mouse, may cause reductions in DNA repair capacity or the fidelity of DNA repair is intriguing; the role of the variants as cancer risk factors or susceptibility alleles remains to be addressed.

Volume-sensitive chloride channels associated with human cervical carcinogenesis.

Previous controversy has risen from the purported equivalence of the volume-sensitive chloride channels with P-glycoprotein. The aim of this study was to investigate the association between expression of volume-sensitive Cl- channels and the process of malignant transformation of cervical epithelial cells. We studied the activations of volume-sensitive and cAMP-mediated chloride currents in various human cervical squamous cells that were representative of different stages of cervical carcinogenesis, i.e., normal cervical epithelium, low-grade cervical intraepithelial neoplasia, carcinoma in situ, and invasive carcinoma using the whole-cell patch clamp technique. The volume-sensitive chloride channels, however, were significantly activated only in the four cervical cancer cell lines, primary culture cells of carcinoma in situ, and invasive cancer of the cervix. The expression of volume-sensitive chloride currents was independent of the state of human papillomavirus positivity. When these cells were exposed to hypotonic shock, the cells swelled, and outward rectified chloride currents were observed. These effects were readily reversed by returning the cells to isotonic medium. In addition, 4,4'-diisothiocyanatostilbene-2,2-disulfonic acid, 1,9-dideoxyforskolin, and verapamil reversibly abolished the volume-sensitive Cl- currents. In contrast, none of the cells from normal cervices and human papillomavirus-immortalized cell lines, the in vitro equivalent of low-grade cervical intraepithelial neoplasia, developed substantial chloride currents on exposure to hypotonicity. cAMP-mediated chloride currents were ubiquitously activated in all cervical squamous cells, regardless of the stages of carcinogenesis. This is the first report suggesting an in vivo association between the development of volume-sensitive chloride currents and human carcinogenesis.

Volume-sensitive chloride channels in the primary culture cells of human cervical carcinoma.

Previous study shows volume-sensitive chloride currents are induced by hypotonicity in human cervical cancer cell lines, but not in normal cervical epithelium. To ascertain whether the preferential activation of these channels in cancer cell lines could be similarly and directly detected in cervical cancer tissues, we studied volume-sensitive chloride channels on the primary culture cells of invasive cervical carcinoma using the whole-cell patch-clamp technique. The process of regulatory volume decrease (RVD) was also studied using electronic cell sizing to measure cell volume. Results demonstrate that, in these cultured cells, RVD was mediated in part by chloride loss through the volume-sensitive Cl- channels. A small background current with a slope conductance of 0.32 +/- 0.07 nS/pF at +30 mV (n=60 cells from 10 different samples) was observed. Hypotonicity induced a fast activating and outward rectifying current which was reversed at about 0 mV, and the slope conductance at +30 mV was increased by 10-fold to 3.62 +/- 0.62 nS/pF. These effects were readily reversed by returning the cells to isotonic medium. Moreover, DIDS, NPPB, and 1,9-dideoxyforskolin, reversibly abolished the volume-sensitive Cl- currents. The EC50 required for the inhibitory effect of DIDS, NPPB and 1,9-dideoxyforskolin was 150, 120, and 50 microM, respectively. Volume-sensitive Cl- channels were ubiquitously expressed in cultured cells from 10 samples of different cancer stages, histopathologic types, and state of HPV DNA positivity. Interestingly, similar outward rectifying chloride currents were activated by intracellular 300 microM GTP gamma S. It is proposed that this Cl- conductance may play an important role leading to RVD in human cervical cancer.

Differential osmosensing signalling pathways and G-protein involvement in human cervical cells with different tumour potential.

Previous studies show that the regulatory volume decrease (RVD) in human cervical cells with different tumour potential may be mediated by different ion channels. The signalling events involved in regulating these channel activities are not clear. To screen the possible mechanisms involved in cell volume regulation in these cells, we examine intracellular mechanisms and second messengers listed as follows: phospholipase C (PLC), phospholipase A2 (PLA2), tyrosine kinase (TK), protein kinase C (PKC), protein kinase A (PKA), and cAMP. The involvement of G-protein was also studied. Our results showed that PLC signalling with downstream activation of PKC was involved in the cell volume regulation of cervical cancer cells. On the other hand, different PKC isoforms that were not related to upstream PLC regulation were involved in the RVD of human papillomavirus (HPV)-immortalised and normal cervical epithelia. Furthermore, GTP-gamma S facilitated the process of RVD in cervical cancer cells, while pertussis toxin retarded this process. In contrast, neither GTP-gamma S nor pertussis toxin showed effect on the RVD responses of HPV-immortalised and normal cervical cells.

Effects of lithium and haloperidol on human sperm motility in-vitro.

Two psychotropic drugs, lithium and haloperidol, were evaluated for their in-vitro effects on sperm motility using a transmembrane migration method. Sperm motility was measured either immediately after semen had been mixed with the drug or after a 2 h incubation period at 37 degrees C. Lithium inhibited human sperm motility in a dose-dependent manner with an EC50 of 10 mM when the semen-lithium mixture had been incubated. Sperm motility was increased to 127% of control when semen had been incubated with 0.027 microM haloperidol; this concentration was within the therapeutic range.

Adenosine stimulates human sperm motility via A2 receptors.

The effects of adenosine and its analogues on human sperm motility were studied using a transmembrane migration method. Specific binding sites for adenosine in human sperm were also investigated. Adenosine and 5'-N-ethylcarboxamidoadenosine (NECA) stimulated human sperm motility with similar efficacies and the maximal amplitudes of motility increases were both about 70%. 3,7-Dimethyl-1-propargylxanthine (DMPX), a potent A2 antagonist, competitively antagonized NECA-induced motility stimulation. Successively higher concentrations of DMPX shifted the dose-response curve of NECA to the right in a nearly parallel fashion. Dipyridamole, an inhibitor of adenosine uptake, does not reduce the ability of adenosine to stimulate human sperm motility. In radioligand-binding studies, adenosine A1 selective analogues, cyclopentyl-1,3-dipropylxanthine and 1-methyl-2-phenylethyl adenosine, have little competitive effect on [3H]NECA binding in human sperm membrane. These results provide evidence that adenosine enhances human sperm motility via adenosine A2 receptors on the surface of sperm membranes.

Calcineurin-mediated dephosphorylation of c-Jun Ser-243 is required for c-Jun protein stability and cell transformation.

The proto-oncogene c-Jun plays an important role in regulating tumor progression. We previously reported that the serine/threonine phosphatase calcineurin (CaN, also called PP2B) dephosphorylates the C-terminus (Ser-243) of c-Jun, resulting in the increase in c-Jun and Sp1 interaction, and subsequent c-Jun-induced gene expression. Here, we demonstrate the interaction of c-Jun and CaN in the nucleus of living cells by fluorescence resonance energy transfer assay and that this interaction is mediated through the calmodulin-binding domain of CaN. Furthermore, c-Jun protein stability was altered by CaN-mediated dephosphorylation at the Ser-243 site of c-Jun. The half-life of the c-Jun mutant, c-Jun-S243A was longer than that of the wild-type c-Jun. Moreover, silencing of endogenous CaN expression led to increased c-Jun ubiquitination and decreased stability. In 46% of clinical cervical tissue samples obtained from patients with cervical cancer, enhanced c-Jun and CaN expression, as well as decreased phospho-Ser-243 expression levels were detected. Our results suggest that CaN stabilizes c-Jun by dephosphorylating c-Jun at Ser-243 to enhance its tumorigenic ability.

Conditionally replicating E1B-deleted adenovirus driven by the squamous cell carcinoma antigen 2 promoter for uterine cervical cancer therapy.

Cervical cancer is the second most common type of malignant tumor among women worldwide. When the disease is confined locally, it can be controlled with surgical resection and radiotherapy. However, patients with recurrent or metastatic disease often have a poor prognosis. Measurement of serum levels of squamous cell carcinoma (SCC) antigens has been widely used as serological markers for SCC of uterine cervix. Recently, it has been demonstrated that cervical cancer patients with elevated squamous cell carcinoma antigen-2 (SCCA2) expression in tumor cells carry a poor prognosis. Here, by using a luciferase reporter assay, we show that SCCA2 promoter was active in SCCA2-producing human cervical cancer cell lines, including Cx, Cxwj, SiHa and HeLa cells, but relatively quiescent in normal cervical epithelial cells. We then developed a conditionally replicating adenovirus, designated Ad-KFH, under the transcriptional control of the SCCA2 promoter. This E1B-55 kDa-deleted oncolytic adenovirus replicated specifically in and lysed SCCA2-producing cervical cancer cells. Furthermore, in a peritoneal metastatic tumor model, Ad-KFH retarded Cxwj tumor growth in NOD/severe combined immunodeficient mice and prolonged survival of tumor-bearing mice, especially when combined with cisplatin. These results suggest that Ad-KFH may provide a new strategy of gene therapy for advanced or recurrent uterine cervical cancer.

Increased expression level of squamous cell carcinoma antigen 2 and 1 ratio is associated with poor prognosis in early-stage uterine cervical cancer.

Squamous cell carcinoma antigen (SCCA) is a tumor marker for patients with squamous cell carcinoma of uterine cervix, lung, and esophagus. It was encoded by two highly homologous genes, SCCA1 and SCCA2. However, the relevance of SCCA genes to squamous cell carcinogenesis and patient outcome remains far from clear. In this study, by using laser microdissection and real-time quantitative polymerase chain reaction procedures, the messenger RNA (mRNA) expression of the SCCA1 and SCCA2 genes in normal, dysplastic, and malignant squamous epithelia from uterine cervical tissues were analyzed and correlated with outcome of cancer patients. We found that the SCCA2/A1 mRNA ratios were progressively increased from normal, dysplastic, to cancer cells, and the mean ratio was significantly higher in cancer tissues than that in normal epithelium (P= 0.02). The SCCA2/A1 mRNA ratios were not significantly associated with types of human papillomavirus infection (P > 0.05). High SCCA2/SCCA1 mRNA ratios (ratio >1) were an independent predictor of disease recurrence (relative risk: 3.58; P= 0.003). Of the 38 patients with cervical cancer, 12 patients with high SCCA2/SCCA1 mRNA ratios had a significant lower 2-year disease-free survival of only 50%, while it was 92% in those with low SCCA2/SCCA1 mRNA ratios (P < 0.001). In conclusion, our study indicated that the ratios of SCCA2 to SCCA1 RNA were increased during the process of cervical carcinogenesis, and patients with elevated SCCA2/A1 ratio carried a higher risk for recurrence in early-stage uterine cervical cancer.

Evidence of human papillomavirus infection, enhanced phosphorylation of retinoblastoma protein, and decreased apoptosis in sarcomatoid squamous cell carcinoma of uterine cervix.

Sarcomatoid squamous cell carcinoma (SSCC) of the uterine cervix, characterized by biphasic components of sarcomatoid and squamous neoplastic cells, is a rare entity with uncertain pathogenesis. To date, less than 20 cases have been mentioned. Although the rarity of this diagnosis makes it difficult to draw firm conclusions from limited data, it does seem that SSCC is an aggressive tumor. In this study, we present a 31-year-old patient with abnormal vaginal bleeding. The diagnosis of SSCC was confirmed through pathologic examinations from hysterectomy specimen. Its epithelial origin was demonstrated by immunohistochemical studies. The expression of p53, HER2/neu, and c-kit was not enhanced in this tumor. Importantly, it was human papillomavirus type 16, positive by polymerase chain reaction and in situ hybridization studies. Enhanced immunostaining for phospho-retinoblastoma protein and decreased apoptosis compared with the squamous cell carcinoma counterpart were observed. This report characterizes the first description of molecular features in SSCC that may account for its aggressive behavior.

An in vivo assay for measuring the recombination potential between DNA sequences in mammalian cells.

Mammalian intermolecular recombination vectors that place the recombination junction within the intron of a selectable marker gene are presented. Many of the previously reported recombination assays require that recombination occur homologously and that they occur within the coding region of the selectable marker. This vector system involves the use of a human thymidine kinase (tk) minigene and measures the recombination frequency between any chosen DNA sequences, in mammalian thymidine kinase negative cells. The tk minigene is divided into a 5' vector and a 3' vector. In the 5' vector, the DNA sequence of interest is inserted in the proximal portion of tk intron 2. In the 3' vector, the DNA sequence of interest is inserted in the intron sequence between the proteolipid protein exon 2 and tk exons 3-7. Recombination through the DNA sequences of interest, either homologous or illegitimate, will reconstruct a functional tk minigene. The recombination junction is spliced out of the transcribed mRNA and thymidine kinase positive cells can be selected in hypoxanthine-aminopterin-thymidine medium. We have tested these vectors to measure the recombination potential of two Alu repetitive sequences (BLUR 8 and BLUR 11) against a control DNA sequence. BLUR 8 and BLUR 11 do not seem to recombine at a significantly higher frequency over that of the control DNA sequence. These recombination vectors display similar sensitivity to previous recombination systems, but allow tremendous flexibility in the choice of potentially recombinogenic sequences.

The inhibitory effect of anticonvulsants on human sperm motility: measured with a trans-membrane migration method.

The in-vitro effects of four anticonvulsant drugs (phenytoin, phenobarbitone, carbamazepine and valproate) on human sperm motility were studied with a trans-membrane migration method. Sperm motility was measured either immediately after semen had been mixed with the drug or after a 2-hour pre-incubation at 37 degrees C. When the drug effect was evaluated after the semen-drug mixture had been pre-incubated for 2 hours, the concentration of phenytoin, carbamazepine and valproate that inhibited sperm motility to 50% of control (EC50) was 1.59, 4.23, and 5.00 mM, respectively. If sperm motility was immediately measured without preincubation, the EC50 was increased to 8.47 and 100.00 mM for carbamazepine and valproate, respectively, whereas phenytoin could not inhibit sperm motility to less than 50% of the control at the tested concentrations. Both with and without pre-incubation, phenobarbitone, even up to 12.92 mM, could not inhibit sperm motility to less than 50% of the control. The result was compatible with previous findings that drugs with a membrane stabilizing effect may inhibit human sperm motility. This study provided further information regarding the application of human sperm motility as a cellular model for future pharmacological research.

BC1 RNA, the transcript from a master gene for ID element amplification, is able to prime its own reverse transcription.

ID elements are short interspersed elements (SINEs) found in high copy number in many rodent genomes. BC1 RNA, an ID-related transcript, is derived from the single copy BC1 RNA gene. The BC1 RNA gene has been shown to be a master gene for ID element amplification in rodent genomes. ID elements are dispersed through a process termed retroposition. The retroposition process involves a number of potential regulatory steps. These regulatory steps may include transcription in the appropriate tissue, transcript stability, priming of the RNA transcript for reverse transcription and integration. This study focuses on priming of the RNA transcript for reverse transcription. BC1 RNA gene transcripts are shown to be able to prime their own reverse transcription in an efficient intramolecular and site-specific fashion. This self-priming ability is a consequence of the secondary structure of the 3'-unique region. The observation that a gene actively amplified throughout rodent evolution makes a RNA capable of efficient self-primed reverse transcription strongly suggests that self-priming is at least one feature establishing the BC1 RNA gene as a master gene for amplification of ID elements.

Evolution of the master Alu gene(s).

A comparison of Alu sequences that comprise more recently amplified Alu subfamilies was made. There are 18 individual diagnostic mutations associated with the different subfamilies. This analysis confirmed that the formation of each subfamily can be explained by the sequential accumulation of mutations relative to the previous subfamily. Polymerase chain reaction amplification of orthologous loci in several primate species allowed us to determine the time of insertion of Alu sequences in individual loci. These data suggest that the vast majority of Alu elements amplified at any given time comprised a single Alu subfamily. We find that, although the individual divergence relative to a consensus sequence correlate reasonably well with sequence age, the diagnostic mutations are a more accurate measure of the age of any individual Alu family member. Our data are consistent with a model in which all Alu family members have been made from a single master gene or from a series of sequential master genes. This master gene(s) accumulated diagnostic base changes, resulting in the amplification of different subfamilies from the master gene at different times in primate evolution. The changes in the master gene(s) probably occurred individually, but their appearance is clearly punctuated. Ten of them have occurred within an approximately 15-million-year time span, 40-25 million years ago, and 8 changes have occurred within the last 5 million years. Surprisingly, no changes appeared in the 20 million years separating these periods.

Volume-sensitive KCI cotransport associated with human cervical carcinogenesis.

This study investigates the volume-sensitive KCI cotransporter (KCC) in various types of human cervical epithelial cell, testing the hypothesis that cervical malignancy is accompanied by differential expression of volume-sensitive KCC. Normal human cervical epithelial cells have KCCs which are quiescent in normal physiological conditions and are relatively refractory to hypotonic stress. By contrast, cervical cancer cells have KCCs which are also nearly quiescent in normal physiological conditions but high transport rates are observed in response to hypotonic challenge. Using isoform-specific primers, mRNA transcripts of KCC1, KCC3 and KCC4 were identified by reverse transcriptase polymerase chain reaction (RT-PCR) in several types of cervical cell, and confirmed by digestion with specific restriction endonucleases. By semiquantitative RT-PCR with beta-actin as the internal standard, the results indicate that cervical carcinogenesis is accompanied by the up-regulation of mRNA transcripts in KCC1, KCC3 and KCC4. [(Dihydroindenyl)oxy] alkanoic acid (DIOA), a KCC inhibitor, blocked both the regulatory volume decrease (RVD) process and volume-sensitive 86Rb+ efflux from cervical cancer cells in a dose-dependent manner. The volume-sensitive 86Rb+ efflux from cervical cancer cells was also blocked by two protein phosphatase inhibitors, calyculin A and okadaic acid, with IC50 values of 0.8 and 6 nM, respectively. Conversely, protein kinase inhibitors, chelerythrine and staurosporine, increased Cl- dependent 86Rb+ efflux. NEM (1 mM) led to a fivefold stimulation of 86Rb+ efflux which was totally Cl- dependent in cervical cancer cells. Hypotonicity could not stimulate any further 86Rb+ efflux after NEM treatment. These results indicate that the volume-sensitive KCC in cervical cancer cells plays a significant role in volume regulation and that the activities are modulated by a phosphorylation cascade. Taken together with our previous studies, we suggest the volume-regulatory ion channels and the co-transport systems work synergistically for volume regulation in human cervical cancer cells.

Swelling-activated taurine and K+ transport in human cervical cancer cells: association with cell cycle progression.

The aim of this study was to investigate swelling-activated taurine and K+ transport in human cervical cancer cells under various culture conditions, testing the hypothesis that the progression of cell cycle was accompanied by differential activities of swelling-activated transport pathways. Aphidicolin, an inhibitor of deoxyribonucleic acid (DNA) synthesis, was used to synchronize the cell cycle. The distribution of cell cycle stage was determined by fluorescence-activated cell sorting (FACS). Hypotonicity activated taurine efflux, which was sensitive to tamoxifen and 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB). Cell swelling also induced both Cl- -dependent and -independent K+ (86Rb+) efflux, presumably mediated by KCl cotransport (KCC) and Ca2+ -activated K+ channels, respectively. Cell cycle arrest in G0/G1 was accompanied by a remarkable decrease in the rate constant for swelling-activated taurine efflux, from 0.20+/-0.007 to 0.026+/-0.002 min(-1) (n=6). The activity of swelling-activated taurine efflux recovered progressively on re-entry into the cell cycle. After removal of aphidicolin and culture with 10% fetal calf serum for 10 h, the rate constant increased significantly from 0.026+/-0.002 to 0.093+/-0.002 min(-1) (n=6). After 24 h release from aphidicolin, the efflux rate constant had increased further to 0.195+/-0.006 min(-1) (n=6), a value not significantly different from that in normally proliferating cells. The differential activities of swelling-activated taurine transport matched well with our previous study showing a volume-sensitive anion channel associated with cell cycle progression. In contrast to the differential activities of swelling-activated taurine transport, swelling-activated K+ (86Rb+) transport was independent of the progression of cell cycle. Most importantly, pharmacological blockade of swelling-activated taurine efflux by tamoxifen or NPPB caused proliferating cervical cancer cells to arrest in G0/G1, suggesting that the activity of this efflux was associated with G1/S checkpoint progression. This study provides new and important information on the functional significance of swelling-activated transport system in the regulation of cell cycle clock of human cervical cancer cells.

Volume-activated taurine transport is differentially activated in human cervical cancer HT-3 cells but not in human papillomavirus-immortalized Z183A and normal cervical epithelial cells.

1. Previous studies demonstrate that volume-sensitive chloride currents are distinctly activated in cervical cancer cells, but not in human papillomavirus (HPV)-immortalized and normal cervical cells. In the present study, the Na(+)-independent volume-activated transport of taurine in three cervical cell types was investigated. 2. Osmotic swelling of cervical cancer HT-3 cells suspended in Na(+)-free hypotonic medium led to increased membrane uptake of taurine. This taurine uptake was effectively blocked by various Cl- channel blockers with a similar potency in blocking volume-sensitive Cl- channels: 1,9-dideoxyforskolin > 5-nitro-2-(3-phenyl-propylamino)-benzoic acid (NPPB) > 4-acetamido-4'-isothiocyanastilbene-2,2'-disulphonic acid (SITS) > 4,4'-diisothio-cyanatostilbene-2,2-disulphonic acid (DIDS) > furosemide. The taurine influx was also abolished by pertussis toxin. In contrast, Na(+)-independent volume-activated taurine transport was not significantly activated in HPV-immortalized Z183A cells and in normal cervical cells. 3. Exposure of HT-3 cells to hypotonic medium also resulted in a marked increase in taurine efflux. The volume-activated taurine efflux was osmolarity dependent and the pattern of pharmacological inhibition by Cl- channel blockers was indistinguishable from that for taurine uptake. 4. These results suggest that volume-sensitive Cl- channels in HT-3 cells can mediate the transport of amino acids. In addition, the pertussis toxin-sensitive G-protein is linked with the activation of this transport mechanism.

Mutations in hamster single-strand break repair gene XRCC1 causing defective DNA repair.

The molecular basis for the DNA repair dysfunction observed in mutant Chinese hamster ovary cell lines of X-ray repair cross complementing group 1 (XRCC1) is unknown and the exact role of the XRCC1 protein remains unclear. To help clarify the role of the XRCC1 gene we analyzed four mutant cell lines of this complementation group and a revertant cell line for XRCC1 protein content and for sequence alterations in the XRCC1 coding region. Immunoblot analysis of cellular extracts indicated that each of four mutant lines was lacking XRCC1 protein, whereas the repair-proficient revertant line derived from one of these mutants contained a normal level of XRCC1. Although each of these cell lines expressed XRCC1 mRNA, we found in all cases a distinct point mutation resulting in crucial alterations in the encoded XRCC1 protein sequence of 633 amino acids. Two of the mutations cause non-conservative amino acid changes, Glu102-->Lys and Cys390-->Tyr, at positions that are invariant among hamster, mouse and human XRCC1 sequences and are located in putative functional domains. A third debilitating mutation disrupts RNA splicing, generating multiple transcripts of different length that contain deletions spanning a region of >100 amino acids in the midsection of the XRCC1 coding sequence. A fourth mutation results in a termination codon that shortens the open reading frame to 220 amino acids, however, in the revertant cell line a further mutation in the same codon, Stop221-->Leu, permits translation of a full-length functional variant protein. These mutational data indicate the importance of the putative functional regions in XRCC1, such as the BRCA1 C-terminal (BRCT) domain found in common with BRCA1 and other DNA repair and cell cycle checkpoint proteins, and also regions necessary for interaction with DNA polymerase beta and DNA ligase III.