Search

The Stroop effect is the delay in reaction times due to interference. Since the original experiments of 1935, it has been used primarily in linguistic context. Language is a complex skill unique to humans, which involves a large part of the cerebral cortex and many subcortical regions. It is perceived primarily in auditory form (spoken) and secondarily in visual form (written), but it is also always perceived in representational form (natural sounds, images, smells, etc). Auditory signals are processed much faster than visual signals, and the language processing centres are closer to the primary auditory cortex than the primary visual cortex, but due to the integration of stimuli and the role of the executive functions, we are able to perceive both simultaneously and coherently. However, auditory signals are still processed faster, and this study focused on establishing how auditory and visual, linguistic and representational stimuli interact with each other and affect reaction times in four Stroop tasks with four archetypal mammals (dog, cat, mouse and pig): a written word against an image, a spoken word against an image, a written word against a natural sound and a spoken word against a natural sound. Four hypotheses were tested: in all tasks reaction times would be faster when the stimuli were congruent (Stroop Hypothesis); reaction times would be faster when both stimuli are auditory than when they are visual (Audiovisual Hypothesis); reaction times would be similar in the tasks where one stimulus is auditory and the other visual (Similarity Hypothesis); finally, reaction times would be slower when stimuli come from two sources than when they come from one source (Attention Hypothesis). Twelve native speakers of Swedish between the ages of 22 and 40 participated. The experiment took place in the EEG lab of the Linguistics Department of Stockholm University. The same researcher (the author) and equipment was used for all participants. The results confirmed the Stroop Hypothesis, did not confirm the Audiovisual and Similarity Hypothesis, and the results of the Attention Hypothesis were mixed. The somewhat controversial results were mostly attributed to a false initial assumption, namely that having two different auditory stimuli (one on each ear) was considered one source of stimuli, and possibly the poor quality of some natural sounds. With this additional consideration, the results seemed to be in accord with previous research. Future research could focus on more efficient ways to test the reaction times of Stroop tasks involving auditory and visual stimuli, as well as different populations, especially neurodiverse and bilingual populations.

Stroop effect is the delay in reaction time that is caused by interference. Differences in perception speed of auditory and visual stimuli, as well as the division of selective attention to more than one sources are combined in five different Stroop tasks in this study, involving three stimuli: a picture, a written word and a heard word. Three hypotheses were formed: reaction times will be faster when stimuli agree; reaction times will be slower as tasks become more complex; reaction times will be different when the target stimulus is auditory or visual. Twenty people participated in all five tasks, which involved one or two linguistic stimuli compared against a picture. The first and third hypotheses showed significant results (p

Nucleotide excision repair (NER) is a multi-enzyme DNA repair pathway in eukaryotes. Several NER genes in this pathway including XPB, XPD, XPA and ERCC-1 have been implicated in anticancer drug resistance in human tumor cells. In this study, we assessed the levels of the above-mentioned proteins in the NCI panel of 60 human tumor cell lines in relation to the cytotoxicity patterns of 170 compounds that constitute the standard agent (SA) database. The database consists of drugs used in the clinic for which a mechanism of action has been at least partially defined. The ERCC-1, XPD and XPB protein expression patterns yielded significant negative Pearson correlations with 13, 32 and 17 out of the 170 compounds, respectively (using p0.05). XPA produced a random assortment of negative and positive correlations, and did not appear to confer an overall resistance or sensitivity to these drugs. Protein expression was also compared with a pre-defined categorization of the standard agents into six mechanism-of-action groups resulting in an inverse association between XPD and alkylating agent sensitivity. Our present data demonstrate that XPD protein levels correlate with resistance to alkylating agents in human tumor cell lines suggesting that XPD is implicated in the development of this resistance. NER activity, using the in vitro cell-free system repair assay, revealed no correlation between NER activity and the level of XPD protein in four cell lines with widely varying XPD protein levels. This lack of correlation may be due to the contribution of XPD to other functions including interactions with the Rad51 repair pathway.

The Oct-4 gene encodes a transcription factor that is specifically expressed in embryonic stem cells and germ cells of the mouse embryo. Cells that differentiate into somatic tissues lose Oct-4 expression. Regulation of Oct-4 gene transcription involves a TATA-less minimal promoter and two upstream elements: the proximal (PE) and distal enhancers (DE). We report here the nucleotide sequence of the 5′ upstream regulatory regions of the human and murine Oct-4 genes. A comparative alignment analysis between these regions and those of the bovine Oct-4 ortholog reveals four conserved regions of homology (CR 1 to 4) between these species (66–94% conservation). The 1A sequence within the mouse PE is located approximately half-way between CR 2 and CR 3. A putative Sp1/Sp3 binding site and the overlapping hormone responsive element (HRE) in CR 1 are identical in all three species. A high number of CCC(A/T)CCC motifs exhibit various levels of homology in these upstream regions. We discuss the importance of these and other sequences and present candidate factors that may bind and regulate Oct-4 gene expression.

Excision repair cross-complementing rodent repair deficiency genes (ERCC) are human genes implicated in nucleotide excision repair. ERCC-2 has been implicated in the repair of DNA damaged by chemotherapeutic agents, and may thus play an important role in anticancer drug resistance. ERCC-2 gene expression is low in primary tumor samples rendering it difficult to quantitate. We have developed a semiquantitative method to measure ERCC-2 gene expression utilizing reverse transcription-polymerase chain reaction (RT-PCR). Total RNA extracted from established human tumor cell lines was reverse-transcribed to obtain cDNA. Serially diluted reference ERCC-2 DNA fragment was amplified by PCR to obtain a 617-bp fragment. A standard curve was then created using densitometry readings of the 617-bp bands on agarose gel. A fixed amount of sample cDNA from each cell line was amplified at the same time and the resultant PCR product was read by densitometer. Using the standard curve, ERCC-2 gene expression in a given amount of total RNA was quantitated and normalized to beta-actin expression. There was minimal variation in three repeated experiments with PCR amplification. ERCC-2 gene expression determined by this semiquantitative PCR was also correlated to ERCC-2 quantitation by Northern blot analysis, with a significant concordance (r = 0.912, P = 0.0002). We also successfully applied this sensitive method to quantify five clinical glioma samples.

Transport of (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU) and (−)-norepinephrine was investigated in SarCNU-sensitive SK-MG-1 and -resistant SKI-1 human glioma cell lines. [ 3 H]SarCNU influx was inhibited by SarCNU, sarcosinamide, and (±)-epinephrine in SK-MG-1 cells with competitive inhibition observed by (±)-epinephrine ( K i = 140 ± 12 μM) and (±)-norepinephrine ( K i = 255 ± 41 μM). No effect on influx was detected in SKI-1 cells. [ 3 H](-)-Norepinephrine influx was linear to 15 sec in both cell lines and temperature dependent only in SK-MG-1 cells. Influx of [ 3 H](−)-norepinephrine was found to be saturable in SK-MG-1 ( K m = 148 ± 28 μM, V max = 1.23 ± 0.18 pmol/μL intracellular water/sec) but not in SKI-1 cells. In SK-MG-1 cells, [ 3 H](−)-norepinephrine influx was found to be inhibited competitively by (−)-epinephrine ( K i = 111 ± 7 μM) and SarCNU ( K i = 1.48 ± 0.22 mM). Ouabain and KCl were able to inhibit the [ 3 H](−)-norepinephrine influx in SK-MG-1 cells, consistent with influx being driven by membrane potential. Several catecholamine uptake 2 inhibitors were able to reduce significantly the influx of [ 3 H](−)-norepinephrine and [ 3 H]SarCNU with no inhibition by a catecholamine uptake 1 inhibitor. These findings suggest that increased sensitivity of SK-MG-1 to SarCNU is secondary to enhanced accumulation of SarCNU mediated via the catecholamine extraneuronal uptake 2 transporter, which is not detectable in SKI-1 cells. The introduction of SarCNU into clinical trials will confirm if increased uptake via the catecholamine extraneuronal uptake 2 transporter will result in increased antitumor activity.

We have previously demonstrated that uptake of the amino acid amide sarcosinamide by the glioma cell line SK-MG-1 occurs via the catecholamine carrier that accommodates epinephrine (Km = 0.284 mM; Vmax = 0.154 nmol/10(6) cells/min). Sarcosinamide chloroethylnitrosourea (SarCNU), a new anticancer agent that exerts increased in vitro antitumor activity against gliomas as compared with BCNU (bis-chloroethylnitrosourea), the standard agent of choice, competitively inhibits sarcosinamide uptake by SK-MG-1 cells [inhibition constant (Ki) = 3.26 mM]. Using radiolabeled N-[3H]-sarcosinamide, we determined the transport of sarcosinamide in HT-29 colon-cancer cells, in Calu-1 lung-cancer cells, and in normal foreskin DHF fibroblasts. Sarcosinamide transport was linear for up to 1 min at 22 degrees C. In HT-29 cells and DHF fibroblasts, the uptake of sarcosinamide followed Michaelis-Menten kinetics of carrier-mediated transport. In HT-29 cells the Michaelis constant (Km) was 2.76 +/- 0.1 mM and the maximal velocity (Vmax) was 2.03 +/- 0.1 nmol/10(6) cells/min, whereas in DHF fibroblasts the respective values were 6.58 +/- 3.90 mM and 12.08 +/- 8.20 nmol/10(6) cells/min. In these two cell lines, neither epinephrine nor leucine significantly reduced sarcosinamide transport. In Calu-1 cells there was no evidence of carrier-mediated transport of either sarcosinamide or epinephrine. These nonglial cell lines lack a high-affinity catecholamine carrier. The increased cytotoxicity of SarCNU in gliomas may correlate with the presence of a high-affinity catecholamine carrier.

Nucleotide excision repair (NER) is a multi-enzyme DNA repair system in eukaryotes. Several NER genes in this system including XPA, XPB, ERCC1, and ERCC2 (XPD) have been implicated in anticancer drug resistance in human tumor cells. This study was designed to investigate the relationship between the expression of NER protein and the drug-resistance of human tumor cell lines.In this study, The authors assessed the levels of the above mentioned proteins, by utilizing Western blot analysis, in the USA National Cancer Institute (NCI) panel of 60 human tumor cell lines and correlated to the cytotoxicity patterns of 170 compounds that constitute the standard agent (SA) database.The ERCC1, XPB, and XPD protein expression patterns yielded significant negative Pearson correlations with 13, 17, and 32 out of the 170 compounds, respectively (P0.05). XPA produced a random assortment of negative and positive correlations and did not appear to confer an overall resistance or sensitivity to these drugs. Protein expression was also compared with a pre-defined categorisation of the standard agents into six mechanism-of-action (MOA) groups resulting in an inverse association between XPD and alkylating agent sensitivity.Our present data demonstrate that XPD protein levels correlate with resistance to alkylating agents in human tumor cell lines, suggesting that XPD plays an important role in the development of this resistance.

We have recently completed screening of the National Cancer Institute human tumor cell line panel and demonstrated that among four nucleotide excision repair proteins (XPA, XPB, XPD, and ERCC1), only the TFIIH subunit XPD endogenous protein levels correlate with alkylating agent drug resistance. In the present study, we extended this work by investigating the biological consequences of XPD overexpression in the human glioma cell line SK-MG-4. Our results indicate that XPD overexpression in SK-MG-4 cells leads to cisplatin resistance without affecting the nucleotide excision repair activity or UV light sensitivity of the cell. In contrast, in SK-MG-4 cells treated with cisplatin, XPD overexpression leads to increased Rad51-related homologous recombinational repair, increased sister chromatid exchanges, and accelerated interstrand cross-link removal. Moreover, we present biochemical evidence of an XPD-Rad51 protein interaction, which is modulated by DNA damage. To our knowledge, this is the first description of functional cross-talk between XPD and Rad51, which leads to bifunctional alkylating agent drug resistance and accelerated removal of interstrand cross-links.

Various mechanisms have been implicated in nitrogen mustard drug resistance. The role of these mechanisms in the development of chlorambucil drug resistance in chronic lymphocytic leukemia (CLL) is discussed. We review these mechanisms with emphasis on the emerging role of DNA repair, and specifically, recombinational repair. Inhibition of these repair processes may lead to new therapies, not only in CLL, but in other malignancies as well.

Adjuvant nitrosourea chemotherapy fails to prolong survival significantly as many tumors demonstrate resistance to these drugs. It has been documented in cell lines that O6-methylguanine DNA methyltransferase (MGMT) plays an important role in chloroethylnitrosourea (CENU) drug resistance.We evaluated MGMT expression in 22 glioma specimens by using an immunofluorescence assay and compared the results with clinical responses of the patients to CENU-based chemotherapy.Eight tumor samples had no detectable MGMT, whereas other samples had from 9,989 to 982,401 molecules/nucleus. In one group (12 patients), the tumor decreased in size or was stable (effective group), whereas in the other group (10 patients), the tumor demonstrated continuous growth during chemotherapy (progressive group). The Mer- patients (MGMT60,000 molecules/nucleus) appeared to have more chance of stable disease or response to CENU therapy than the Mer+ patients (MGMT60,000 molecules/nucleus) (X2 = 4.791, p = 0.0286). In patients with glioblastomas multiforme (GBMs), the median time to progression (TTP) of Mer+ patient was shorter than that of Mer- patient (t = 2.04, p = 0.049). As a corollary, the MGMT levels were significantly higher in GBM tumors from the progressive group than those from the effective group (t = 2.26, p = 0.029). However, there was no significant correlation between MGMT levels and either the survival time (r = 0.04, p = 0.8595) or TTP (r = 0.107, p = 0.6444).This study suggests that being MGMT positive is indicative of a more aggressive disease that progresses more rapidly with CENU therapy. However, MGMT negative tumors are not always sensitive to CENU agents, suggesting that other factors are also important.

We examined the O6-methylguanine-DNA methyltransferase (MGMT) protein as well as MGMT activity levels and the excision repair cross-complementing rodent repair deficiency gene, ERCC2 (XPD), protein levels in 14 human tumor cell lines not selected for chloroethylnitrosourea (CENU) resistance. These results were compared with 1,3-bis-(2-chloroethyl)-1-nitrosourea (BCNU) cytotoxicity and UV light sensitivity. MGMT protein correlated significantly with MGMT activity (r = 0.9497, p = 0.0001). There was no significant linear correlation between BCNU cytotoxicity and MGMT content as determined by both Western analysis (r = 0.139, p = 0. 6348) and activity assay (r = 0.131, p = 0.6515). However, MGMT-rich cell lines were found to be more resistant than MGMT-poor cell lines to BCNU (t = 2.2375, p = 0.0225) but not to UV (t = 1.1734, p = 0.1317). Furthermore, the most BCNU-sensitive cell lines were all MGMT-poor. UV sensitivity was significantly correlated to BCNU cytotoxicity (r = 0.858, p = 0.0001). Significant correlations were found between ERCC2 protein levels and BCNU cytotoxicity (r = 0.786, p = 0.0009) or UV sensitivity (r = 0.874, p = 0.0001). Our results confirm that MGMT plays an important role in CENU resistance, but not in UV resistance. The correlation of UV sensitivity with BCNU cytotoxicity suggests that nucleotide excision repair is an important modifying factor of MGMT-mediated innate CENU resistance in human tumor cell lines, especially in highly resistant cell lines. ERCC2 may be implicated in this process.

We have developed a method to quantitate ERCC-2 gene expression in tumor cell lines. A mutant ERCC-2 DNA fragment (1-bp mutation) is used as a competitive DNA template in a coamplification PCR reaction with cDNA obtained by reverse transcribing DNase-free total RNA from six human tumor cell lines. The PCR products are separated on agarose gel by virtue of their differential banding pattern upon restriction enzyme digestion. Densitometric readings of the PCR products from a negative film of the gel are used to establish a linear regression curve, which in turn is used to quantitate ERCC-2 levels. Beta-actin expression is similarly quantitated. Normalized ERCC-2 gene expression (either to beta-actin or to total RNA) correlates with cytotoxicity of 1,3-bis-(2-chloroethyl)-1-nitrosourea or (2-chloroethyl)-3-sarcosinamide-1-nitrosourea, suggesting that ERCC-2 may play an important role in drug resistance in these cell lines. This method is reliable and can be used to quantitate gene expression in clinical tumor specimens.

SKI-1 is a 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU)-resistant glioma cell line and SK-MG-1 is a BCNU-sensitive glioma cell line. Both cell lines do not express O6-methylguanine-DNA methyl transferase (MGMT) and exhibit comparable levels of 3-methyladenine DNA glycosylase. In order to detect DNA binding proteins involved in alternative DNA repair mechanisms of BCNU damage, we performed Southwestern analysis using a DNA probe damaged with BCNU and nuclear protein extracts from SKI-1 and SK-MG-1 cell lines. Both cell lines express a protein of M(r) 116,000 that is able to bind to BCNU-damaged DNA with higher specificity than to undamaged DNA. This protein was identified as poly(ADP-ribose) polymerase (PARP). Using glioma extracts depleted of PARP or using antibody to block the DNA binding domain of PARP no other protein binding to BCNU-treated probe was observed. Addition of methoxyamine, an inhibitor of DNA strand breaks, led to a significant reduction of PARP binding to BCNU-treated DNA. BCNU treatment of both glioma cell lines led to reduced nicotinamide adenine dinucleotide levels, indicating activation of PARP. Thus, the recognition and binding of PARP to BCNU-induced DNA nicks with concomitant PARP activation may be important processes that are involved in the initial stage of DNA repair of BCNU lesions in glial cells.

Resistance to (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU), an experimental anticancer compound, was investigated in the chloroethylnitrosourea-sensitive Mer- SK-MG-1 and -resistant Mer- SKI-1 human glioma cell lines. The transport of [3H]SarCNU was examined in suspension. The uptake of [3H]SarCNU was found to be temperature dependent in SK-MG-1 and SKI-1, but less so in SKI-1. At 37 degrees C, uptake of 50 microM [3H]SarCNU was linear up to 4 s in both cell lines, with uptake being significantly faster in SK-MG-1 than in SKI-1 under initial rate conditions. There was no significant difference in the rate of influx at 22 degrees C between both cell lines. Equilibrium was approached after 1 min at 22 and 37 degrees C. At 37 degrees C, steady state accumulation of SarCNU at 30 min was reduced significantly (35%) in SKI-1 cells compared with SK-MG-1 cells, although accumulation was similar at 22 degrees C. In SK-MG-1 cells, uptake of [3H]SarCNU at 37 degrees C was found to be saturable, but uptake in SKI-1 cells was not saturable over a 1000-fold range of concentrations. Analysis of efflux in cells preloaded with 50 microM [3H]SarCNU revealed that the rate of efflux was equivalent in both cell lines but that the efflux rate was more rapid at 37 degrees C compared with 22 degrees C. Metabolism of SarCNU at 37 degrees C was not different in either cell line after a 60-min incubation, as determined by thin layer chromatography. SKI-1 cells, compared with SK-MG-1 cells, were 3-fold more resistant to SarCNU at 37 degrees C but only 2-fold more resistant at 22 degrees C, a temperature at which SarCNU accumulation was similar in both cell lines. The 2-fold resistance at 22 degrees C was similar to that of 1,3-bis(2-chloroethyl)-1-nitrosourea at 37 and 22 degrees C. These findings indicate that increased cytotoxicity in SK-MG-1 cells is associated with a greater accumulation of SarCNU via an epinephrine-sensitive carrier that is not detectable in SKI-1 cells. However, part of the chloroethylnitrosourea resistance in SKI-1 cells is not secondary to decreased accumulation.

As a means of identifying damage recognition proteins involved in repair of nitrogen mustard lesions in chronic lymphocytic leukemia, we performed Southwestern analysis using a probe damaged with melphalan and protein extracts from chronic lymphocytic leukemia patients. We detected proteins with molecular weights of 116,000, 66,000, and 64,000 which bound the damaged probe with a higher specificity than the undamaged probe. The M(r) 66,000 and 64,000 proteins were determined to be degradation products of the M(r) 116,000 protein. The M(r) 116,000 protein was identified as poly(ADP-ribose) polymerase. The use of methoxyamine, an inhibitor of DNA strand breakage following depurination, significantly reduced binding of the melphalan damaged probe to poly(ADP-ribose) polymerase. Following depletion of poly(ADP-ribose) polymerase from the cell extracts, no other binding activity was discovered. Thus, poly(ADP-ribose) polymerase is the only demonstrable protein in chronic lymphocytic leukemia cells which can bind to a DNA probe damaged with melphalan.

The transport of (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU), an experimental anticancer compound, was investigated in the human glioma cell line SK-MG-1. The transport of [3H]SarCNU was examined in suspension. The uptake of [3H] SarCNU was found to be temperature dependent, with influx being linear to 4 sec at 37 degrees. Equilibrium was reached after 1 min at 22 degrees and 37 degrees, with accumulation slightly above unity. SarCNU was not significantly metabolized in the cells after a 60-min incubation at 37 degrees, as shown by thin layer chromatography. At 37 degrees, uptake of [3H]SarCNU was found to be saturable, sodium independent, and energy independent. Previous work demonstrated that SarCNU was able to inhibit the uptake of sarcosinamide, which is transported by the catecholamine uptake 2 system. This catecholamine system mediates the physiological transport of epinephrine. Epinephrine was able to significantly inhibit the uptake of [3H]SarCNU, at a concentration of 50 microM, by 40%. Additionally, several amino acids were unable to inhibit the uptake of SarCNU. The initial rate of SarCNU influx is mediated by both facilitated and nonfacilitated diffusion. The nonfacilitated diffusion rate could be estimated from the linear concentration dependence of the residual influx rate for SarCNU, which was not inhibited by the presence of excess co-permeant (epinephrine). Dixon plot analysis, corrected for nonfacilitated diffusion of SarCNU, revealed that epinephrine inhibited the uptake of SarCNU competitively, with a Ki of 163 +/- 15 microM, a value similar to the Km value for epinephrine influx in SK-MG-1 cells. Additionally, after appropriate corrections for nonfacilitated diffusion in the influx rates observed for SarCNU, it was revealed that SarCNU influx obeyed Michaelis-Menten kinetics over a 200-fold range of concentrations, with a Km of 2.39 +/- 0.37 mM and a Vmax of 236 +/- 53 pmol/microliters of intracellular water/sec. Metabolic poisons (2,4-dinitrophenol, iodoacetate, NaCN, NaF, or ouabain) were unable to inhibit the influx of SarCNU, suggesting that the carrier-mediated uptake of SarCNU is energy independent and mediated by facilitated diffusion. These findings indicate that SarCNU uptake in SK-MG-1 cells is mediated both by nonfacilitated diffusion and by facilitated diffusion via the catecholamine uptake 2 carrier system. SarCNU is the first chloroethylnitrosourea that has been demonstrated to have carrier-mediated uptake. Moreover, this carrier-mediated uptake may play a role in the increased cytotoxicity of SarCNU against gliomas, compared with that of 1,3-bis(2,-chloroethyl)-1-nitrosourea, which enters cells primarily by passive diffusion.

Resistance to the nitrogen mustards in patients with chronic lymphocytic leukemia (CLL) correlates with an enhanced removal of melphalan-induced DNA interstrand cross-links. This finding suggests that DNA repair enzymes may be involved in this process. The activity of 3-methyladenine-DNA glycosylase, which can release altered bases, including adducts at the N-7 position of guanine, was increased significantly in lymphocytes from patients with resistant CLL compared with those from untreated CLL patients. Since glycosylase activity varies with cell proliferation, the amount of [3H]thymidine incorporated into DNA was determined and found to be elevated almost threefold in lymphocytes from patients with resistant CLL. The ratio of glycosylase activity to level of thymidine incorporation did not differ between these two groups of patients. Northern blot analysis of ERCC1 gene (a putative DNA repair enzyme involved in nucleotide excision repair) expression in lymphocytes from patients with CLL revealed multiple gene transcripts (1.1, 3.4, and 3.8 kilobases). In addition, analysis of two samples revealed the presence of a 2.6-kilobase transcript. The 2.6-kilobase transcript was recognized by specific RNA probes that hybridize to antisense ERCC1 transcripts. Levels of expression of the 1.1-kilobase protein encoding transcript in lymphocytes from patients with resistant CLL were increased twofold to threefold above those of untreated patients with CLL. These results indicate that increased expression of ERCC1 and increased activity of 3-methyladenine-DNA glycosylase occur with the development of resistance to the nitrogen mustards in patients with CLL, suggesting a role for enhanced DNA repair in this process.