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Biomedical subjects

L C Panasci

Publications and source records attributed to L C Panasci.

At least 37 records · Page 2Linked to original sources
PubMed1996

Correlation of chloroethylnitrosourea resistance with ERCC-2 expression in human tumor cell lines as determined by quantitative competitive polymerase chain reaction.

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.

Antineoplastic Agents↗
PubMed1996

Identification of a 116 kDa protein able to bind 1,3-bis(2-chloroethyl)-1-nitrosourea-damaged DNA as poly(ADP-ribose) polymerase.

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.

Antineoplastic Agents, Alkylating↗
PubMed1995

DNA repair enzyme expression in chronic lymphocytic leukemia vis-à-vis nitrogen mustard drug resistance.

Nitrogen mustard resistance in B-cell chronic lymphocytic leukemia (B-CLL) has been associated with enhanced DNA repair and increased expression of DNA repair enzymes. Lymphocytes from patients with nitrogen mustard resistant B-CLL displayed a fivefold increase in resistance to melphalan in vitro as compared to those from untreated patients concordant with our definition of clinical resistance. We have performed Northern analysis using a cohort consisting of 11 untreated and 12 treated-resistant patients. Increased expression of ERCC-1 was not found to be associated with nitrogen mustard resistance, nor did we find altered expression of the DNA repair enzymes: ERCC-2, DNA polymerase beta, or topoisomerase I. There was also no difference in the levels of ERCC-1 protein between melphalan sensitive and resistant B-CLL lymphocytes. Analysis of genes involved in nitrogen mustard detoxification revealed that metallothionein was weakly expressed, while transcripts encoding glutathione-S-transferase alpha were undetectable. Thus, it is unlikely either of these proteins plays a role in the resistance. The results of the cytotoxicity assay validate the use of B-CLL as a model to study nitrogen mustard resistance. This model allows us to perform in vitro studies using a tumor which develops resistance in vivo. The results of this study suggest that nucleotide excision repair, as represented by ERCC-1 and ERCC-2, is not the limiting step in B-CLL nitrogen mustard resistance.

Aged↗
PubMed1995

Nitrogen mustard drug resistant B-cell chronic lymphocytic leukemia as an in vivo model for crosslinking agent resistance.

Acquired resistance is a limiting factor in chemotherapy. We have employed nitrogen mustard resistant B-cell chronic lymphocytic leukemia (B-CLL) as a clinically relevant model to study this phenomenon. Resistance in B-CLL is associated with enhanced repair of nitrogen mustard crosslinks. In order to identify the repair pathway responsible for nitrogen mustard resistance, lymphocytes were screened for cross-resistance to a variety of DNA damaging agents. The MTT assay was used to measure the resistance of B-CLL lymphocytes to various DNA damaging agents, including nitrogen mustards, UV light, methyl methanesulfonate, and mitomycin C. We have shown that B lymphocytes from patients with nitrogen mustard resistant chronic lymphocytic leukemia reflect their clinical status. This assay allows us to classify lymphocytes as nitrogen mustard sensitive or resistant, based on in vitro observations. The resistant population was 5.6 and 4.1 fold more resistant to the nitrogen mustard analogs, chlorambucil and melphalan, respectively. Resistant lymphocytes displayed no increased resistance to either methyl methanesulfonate or UV light, indicating that neither classical base nor nucleotide excision repair is rate-limiting in resistance. Resistant lymphocytes were 6.0 and 2.2 fold more resistant to mitomycin C and cis-diamminedichloroplatinum (II), respectively, suggesting enhanced crosslink repair. Neither glutathione nor glutathione S-transferase levels correlated with resistance. The development of nitrogen mustard drug resistance in B-CLL appears to be associated with cross-resistance to other bifunctional alkylating agents which produce interstrand crosslinks. Our results indicate that resistance to nitrogen mustards in chronic lymphocytic leukemia is associated with enhanced repair of DNA crosslinks which may involve a recombination dependent system. This model should prove very useful in the elucidation of the molecular mechanisms of crosslink repair.

B-Lymphocytes↗
PubMed1994

Altered cytotoxicity of (2-chloroethyl)-3-sarcosinamide-1-nitrosourea in human glioma cell lines SK-MG-1 and SKI-1 correlates with differential transport kinetics.

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.

Antineoplastic Agents↗
PubMed1993

A successful system of scientific data audits for clinical trials. A report from the Cancer and Leukemia Group B.

OBJECTIVE: To report on data collected during on-site audits of source documents in the Cancer and Leukemia Group B (CALGB). DESIGN: A retrospective review of audit reports in four audit cycles. SETTING: A cooperative group of institutions conducting clinical trials in cancer treatment. PARTICIPANTS: Patients taking part in clinical trials at collaborating CALGB institutions, members of the CALGB Data Audit Committee, and group chairmen of CALGB. MAIN OUTCOME MEASURE: The results of 691 institutional audits conducted by the CALGB in 1982 through 1992 with comparisons of main CALGB institutions vs affiliates. RESULTS: In four full reviews of all participating institutions in the CALGB, 3787 patients have had their on-site medical records compared with data submitted to the CALGB Data Management Center. Compliance with federal regulations for oversight by an institutional review board improved from a deficiency rate of 28.0% among the main institutions and 49.6% of the affiliate institutions in the first audit cycle to respective figures of 13.3% and 28.2% in the fourth cycle. Consent form deficiencies also dropped overall from 18.5% in the first cycle to 3.9% in the fourth. Patient eligibility was verified by auditors in 94.5%, and assessment of tumor changes in response to treatment was verified in 96.4% in the fourth cycle; both figures were only slightly lower in the first cycle. Two instances of scientific impropriety were discovered for a rate of only 0.28% of all audits. Both occurred prior to 1984, and none have occurred since. Major protocol deviations in drug dosing have held steady at about 11% over four audit cycles. Over the 11-year period of audits, three main institutions and 96 affiliate institutions have discontinued CALGB membership due solely, or at least partly, to unfavorable audit results. CONCLUSION: Scientific improprieties have occurred very rarely in clinical trials conducted by the CALGB. Protocol compliance in assessing patient eligibility and tumor responses has been high. Attention to administrative matters of consent forms, institutional review board approval, and ancillary data submission has measurably improved in the CALGB, which is at least partly due to the pressure from this on-site peer review of investigator performance.

Clinical Trials as Topic↗
PubMed1993

Effect of ERCC-1 overexpression on sensitivity of Chinese hamster ovary cells to DNA damaging agents.

Previous studies of ERCC-1 gene expression levels in chronic lymphocytic leukemia and ovarian carcinoma tumor specimens indicated that increased gene expression may correlate with a lack of response to the alkylating agents, melphalan, and cis-diamminedichloroplatinum (II). In order to demonstrate direct involvement of the ERCC-1 protein in repair of melphalan lesions, the ERCC-1 defective Chinese hamster ovary cell line UV20 was transfected with the human ERCC-1 complementary DNA. Stably transfected UV20 cells demonstrated an increase in resistance to melphalan. Wild type Chinese hamster ovary AA8 cells were then stably transfected with the same complementary DNA. The result was an increase in sensitivity to melphalan. There was no effect on sensitivity to uv light, but the ERCC-1 transfected AA8 cells had an increased sensitivity to cis-diamminedichloroplatinum (II). These results suggest that overexpression of human ERCC-1 may inhibit a pathway specific to the repair of bifunctional DNA damaging agent lesions in AA8 cells. ERCC-1 transfected AA8 cells should be useful in determining the precise role of ERCC-1 in repair of DNA cross-links induced by melphalan.

Animals↗
PubMed1993

Transport of (2-chloroethyl)-3-sarcosinamide-1-nitrosourea in the human glioma cell line SK-MG-1 is mediated by an epinephrine-sensitive carrier system.

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.

Antineoplastic Agents↗
PubMed1992

Lack of evidence for a high-affinity sarcosinamide carrier or a catecholamine carrier in Calu-1 lung-cancer cells, HT-29 colon-cancer cells, and DHF fibroblasts.

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.

Biological Transport↗
PubMed1990

Transport of amino acid amide sarcosinamide and sarcosinamide chloroethylnitrosourea in human glioma SK-MG-1 cells.

The transport of the amino acid amide N-[3H]sarcosinamide (methyl glycinamide) was investigated in human glioma SK-MG-1 cells. Sarcosinamide uptake was found to be temperature dependent, sodium independent, and linear up to 1 min at 22 degrees C. Equilibrium was reached after 10 min at 22 degrees C with accumulation slightly above unity. Sarcosinamide was not metabolized in the cells as shown by thin layer chromatography. The uptake of sarcosinamide was significantly decreased when the extracellular pH was lowered from 7.5 to 6.0 and significantly enhanced at pH values above 7.5. The latter effect may be due mainly to increased cell permeability at high pH. The uptake of the labeled sarcosinamide was trans-stimulated by excess cold sarcosinamide. Sarcosinamide uptake over a 200-fold range of concentrations followed Michaelis-Menten kinetics with a Km of 0.284 +/- 0.041 mM and a Vmax of 0.154 +/- 0.024 nmol/10(6) cells/min. The uptake of sarcosinamide was significantly reduced by iodoacetate but not by the metabolic poisons NaF, ouabain, or dinitrophenyl, suggesting that the uptake is not dependent on energy, rather it proceeds by facilitated diffusion. Several naturally occurring substrates were unable to inhibit the uptake of sarcosinamide. Leucine significantly reduced the uptake of sarcosinamide, while sarcosinamide was a weak inhibitor of leucine transport. 2-Aminobicyclo[2,2,1]heptane-2-carboxylic acid a specific substrate for the sodium-independent, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid-sensitive amino acid system L failed to inhibit the uptake of sarcosinamide. Epinephrine reduced the uptake of sarcosinamide and sarcosinamide was equally potent as an inhibitor of epinephrine transport. Dixon plot analysis demonstrated that epinephrine (Km = 0.270 mM) inhibits the uptake of sarcosinamide competitively (Ki = 0.260 mM). These results indicate that sarcosinamide is a substrate for the catecholamine transporter. The alkylating agent, sarcosinamide chloroethylnitrosourea, was tested for its ability to inhibit the uptake of sarcosinamide. The results of Dixon plot analysis were consistent with competitive inhibition of sarcosinamide uptake and the inhibition constant Ki for SarCNU was found to be 3.26 +/- 0.57 mM. The steady-state intracellular concentration of SarCNU was found to be significantly higher (cell:medium ratio of 1.03 +/- 0.01) than that of BCNU cell:medium ratio of 0.52 +/- 0.12). These findings indicate that SarCNU and sarcosinamide share the same carrier for uptake in SK-MG-1 cells. This transport mechanism may be responsible for the increased accumulation of SarCNU as compared to BCNU, a nitrosourea which enters cells by passive diffusion.

Biological Transport, Active↗
PubMed1990

Statin expression in the untreated and SarCNU-exposed human glioma cell line, SK-MG-1.

Cytokinetic analyses of gliomas and other neoplasms rely exclusively on the use of proliferation-dependent markers such as [3H]-thymidine and BuDR incorporation and the detection of growth-dependent proteins such as proliferating cell nuclear antigen (PCNA) and Ki-67. In normal tissues, the monoclonal antibody S-44 recognizes statin, a nuclear protein expressed only in nonproliferating cells. In the present study, indirect immunofluorescence microscopy using S-44 identified nuclear statin in 5.9% of a population of untreated human SK-MG-1 glioma cells in vitro. Incremental doses of the alkylating agent sarcosinamide chloroethylnitrosourea (SarCNU) induced a linear increase in the fraction of statin-positive SK-MG-1 cells. Labeling of nuclear statin with the monoclonal antibody S-44 may be a potentially useful marker of the cytotoxic effects of anticancer drugs in gliomas and other neoplastic tissues.

Antibodies, Monoclonal↗
PubMed1990

Mechanisms of resistance to (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU) in sensitive and resistant human glioma cells.

Resistance to (2-chloroethyl)-3-sarcosinamide-1-nitrosourea (SarCNU), an experimental antitumor compound, was investigated in the sensitive SK-MG-1 cells and the 20-fold more resistant SKI-1 human glioma cells [which are 3-fold more resistant to 1,3,bis(2-chloroethyl)-1-nitrosourea (BCNU)]. The transport of SarCNU was examined by utilizing tritiated sarcosinamide. Sarcosinamide uptake into SK-MG-1 cells is via the catecholamine carrier that accommodates epinephrine. Dixon plot analysis of SarCNU inhibition of sarcosinamide uptake reveals that SarCNU is also accommodated by this carrier. The uptake of 0.5 mM [3H]sarcosinamide was temperature dependent, with similar levels of intracellular sarcosinamide accumulating at steady state in both cell lines. The uptake of sarcosinamide in SKI-1 cells obeyed Michaelis-Menten kinetics over a 200-fold range of concentrations with a Km of 1.52 +/- 0.151 mM and Vmax of 0.659 +/- 0.066 nmol/10(6) cells/min. This represents a more than 5-fold decrease in the uptake affinity and a more than 4-fold increase in the transport capacity compared with SK-MG-1 cells (Km = 0.282 +/- 0.041 mM; Vmax = 0.154 +/- 0.024 nmol/10(6) cells/min). The initial rate of sarcosinamide uptake is similar in both cell lines. Dixon plot analysis confirmed that SarCNU is a competitive inhibitor of sarcosinamide transport in SKI-1 cells with a Ki of 17.5 mM, which is more than 5-fold greater than the Ki obtained in SK-MG-1 cells. The steady state accumulation of SarCNU is significantly reduced by 47% in SKI-1 cells compared with the SK-MG-1 cells (cell to medium ratios of 0.65 +/- 0.11 and 1.22 +/- 0.08, respectively) (p less than 0.005). The accumulation of BCNU was comparable in the two cell lines. Since the Vmax of sarcosinamide (SarCNU) uptake is increased in the SKI-1 cells, the decrease in intracellular SarCNU is not related to decreased drug influx via the catecholamine carrier in SKI-1 cells. The efflux of tritiated sarcosinamide was temperature dependent and similar in both cell lines, with 54 and 58% of sarcosinamide being freely exchangeable in SKI-1 and SK-MG-1 cells, respectively. SarCNU efflux may or may not be altered. Since the expression of mdr is higher in the sensitive cells, it is unlikely that increased efflux of SarCNU mediated by the P-glycoprotein is responsible for drug resistance.(ABSTRACT TRUNCATED AT 250 WORDS)

Acetyltransferases↗
PubMed1989

Evidence for increased alpha MSH receptor binding in the F1 variant of B16 melanoma cells grown in dialyzed fetal calf serum.

The specific binding of an alpha MSH analogue (Ac-[Nle4, D-Phe7] alpha MSH4-11 NH2) was enhanced in the presence of 10% dialyzed fetal calf serum (FCS) as compared with 10% FCS (nondialyzed) in the F1 variant of B16 melanoma cells. The replenishment of dialyzed serum with adrenocorticotrophic hormone (ACTH) or insulin had no effect on the increased level of alpha MSH receptor binding in these cells. However, 10 nM alpha MSH or 1 microM ACTH under identical conditions significantly decreased the level of alpha MSH binding. Competitive binding studies involving the alpha MSH analogue revealed that the specificity of the receptor was restricted to the complete molecule of alpha MSH, our analogue, beta MSH and ACTH1-24, ACTH4-10, which contains the amino acid sequence responsible for biological activity, showed a very low affinity for the receptor. Furthermore, we observed an interesting phenomenon unique to dialyzed FCS in that once the cells were grown to confluence and melanin was produced, the cells were no longer viable. However, in McCoy's medium, which is deficient in tyrosine, the cells did not produce melanin and remained viable.

Animals↗
PubMed1989

The cytotoxicity of a 2-chloroethylnitrosourea analog of sarcosinamide in the SK-MG-1 human glioma cell line as a possible indicator for transport.

The cytotoxicities of a new sarcosinamide analog of chloroethylnitrosourea (SarCNU) and of BCNU were examined in the glioma cell line SK-MG-1 in the presence or absence of excess concentrations of amino acids and sarcosinamide. The cytotoxicity of SarCNU, but not of BCNU, was significantly reduced in the presence of excess sarcosinamide. The stability of SarCNU was not significantly altered by increasing concentrations of sarcosinamide. In order to investigate the possibility that sarcosinamide inhibits the uptake of SarCNU the transport of tritiated sarcosinamide was examined in SK-MG-1 cells. The uptake of 3H-sarcosinamide was inhibited by excess, unlabelled sarcosinamide and SarCNU but not by BCNU, glycine or sarcosine. These results suggest the existence of a carrier-mediated transport for sarcosinamide which can accomodate SarCNU in SK-MG-1 cells.

Antineoplastic Agents↗
PubMed1987

Mechanism of decrease of protein synthesis by sodium cyanate in murine P388 leukemia cells.

Sodium cyanate is a selective in vivo inhibitor of protein synthesis in a variety of mammalian tumor cells without a corresponding effect on the normal tissues of tumor-bearing animals. The in vivo decrease of protein synthesis observed 4 h post-NaOCN i.p. administration in the murine P388 leukemia cell cannot be explained by decreased amino acid pools in the mouse peritoneal cavity. In addition, the decrease in protein synthesis observed with NaOCN in isolated P388 cells was shown not to be secondary to (a) alterations in the kinetics of amino acid transport or (b) effects on total nucleotide pools. The incorporation of [14C]phenylalanine in P388 cell-free lysates from NaOCN-pretreated mice was significantly decreased to approximately 55% of control lysates in the presence of exogenous amino acids. The addition of exogenous calf liver tRNA to the lysates did not alter this result. However, no difference was observed in polyuridylic acid-directed [14C]phenylalanine incorporation into polypeptides in micrococcal nuclease-treated P388 lysates from NaOCN-pretreated or control mice. Quaternary initiation complex (48S) formation and mRNA synthesis were found to be significantly decreased by 35 and 38%, respectively, in P388 cells from NaOCN-pretreated mice. DNA synthesis was decreased by 66% of control at 1 h and 62% at 4 h post-NaOCN i.p. administration. No apparent effect with NaOCN was observed on total RNA synthesis in P388 cells. These results suggest that the decrease in P388 cell protein synthesis observed with NaOCN in vivo appears to be due to alterations manifested in the synthesis of cellular mRNA and protein synthesis initiation processes. NaOCN does not appear to affect the P388 cell ribosomal machinery, tRNA, or protein synthesis elongation processes.

Adenosine Triphosphate↗
PubMed1987

Further studies on amino acid transport in murine P388 leukemia cells in vitro. Presence of system y+.

The transport of glycine and L-lysine into murine P388 leukemia cells has been examined. Glycine transport appears to be shared by both systems A and ASC in P388 cells. Glycine transport is Na+-dependent and is effectively blocked by alpha-(methylamino)isobutyric acid, threonine and alanine but only a marginal reduction in transport is seen with 100-fold excess cold 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid. System gly is not expressed in P388 cells. Lysine is largely transported by a Na+-independent, pH-insensitive system with a Km of 0.079 mM. Lysine transport is relatively unaffected by the addition of 100-fold excess cold alpha-(methylamino)isobutyric acid, 2-aminobicyclo[2,2,1]heptane-2-carboxylic acid and the anionic amino acids, L-glutamate and L-aspartate. A partial inhibition of lysine transport was observed with L-threonine and L-leucine while L-arginine and L-histidine radically decreased lysine transport. Lysine appears to be transported by a system similar to the system y+ seen in cultured human fibroblasts, Ehrlich ascites cells, and hepatoma cell lines.

Amino Acids↗