Antiproliferative effects of 9-beta-D-arabinofuranosyladenine 5'-monophosphate and related compounds in combination with adenosine deaminase inhibitors against mouse leukemia L1210/C2 cells in culture.

The antiproliferative activity of 9-beta-D-arabinofuranosyladenine 5'-monophosphate against a cultured line of mouse leukemia cells (L1210/C2) was enhanced by addition of either 2'-deoxycoformycin or erythro-9-(2-hydroxy-3-nonyl)adenine. The activity of 9-beta-D-arabinofuranosyladenine 5'-monophosphate, alone or in combination with either of the two inhibitors of adenosine deaminase, was comparable to that of 9-beta-D-arabinofuranosyladenine (ara-A), apparently reflecting the rapid conversion of 9-beta-D-arabinofuranosyladenine 5'-monophosphate to ara-A by L1210/C2 cells. Several ara-A analogs were assayed for antiproliferative activity against L1210/C2 cells; of these, only 9-beta-D-arabinofuranosyladenine 5'-O-methylphosphate and 2'-deoxy-2'-amino-9-beta-D-arabinofuraosyladenine were active. Addition of 2'-deoxycoformycin to cell culture fluids enhanced the activity of 9-beta-D-arabinofuranosyladenine 5'-O-methylphosphate suggesting conversion to an adenosine deaminase-sensitive intermediate, presumably ara-A.

Resistance to 9-beta-D-arabinofuranosyladenine in cultured leukemia L 1210 cells.

A cultured line of L1210 leukemia cells, designated L1210/ara-A, was selected for resistance to 9-beta-D-arabinofuranosyladenine (ara-A) by a series of 72-hr exposures to increasing concentrations of ara-A in the presence of 1 microM deoxycoformycin. Cells of the resistant line were about one-tenth as sensitive as were cells of the parent line to the effects of ara-A on proliferation, viability, and tumorigenicity. Cross-resistance, as determined by comparison of drug effects on rates of proliferation of L1210/C2 and L1210/ara-A cells, was seen with adenosine, deoxyadenosine, methylmercaptopurine ribonucleoside, tubercidin, and cordycepin but not with 1-beta-D-arabinofuranosylcytosine or with 9-beta-D-arabinofuranosyl-2-fluoroadenine. The levels of resistance to methylmercaptopurine ribonucleoside, cordycepin, and tubercidin were considerably greater than that seen with ara-A itself. L1210/C2 and L1210/ara-A cells were compared with respect to the effects of ara-A on cell size distributions, DNA distributions, labeling indices, and apparent rates of DNA synthesis, and the differences seen were consistent with inhibition of DNA synthesis and unbalanced growth as the major mechanism of ara-A cytotoxicity. The decreased sensitivity of DNA synthesis in L1210/ara-A cells treated with ara-A, relative to L1210/C2 cells, was due to reduced intracellular accumulation of ara-A phosphates in the resistant line. Phosphorylation of ara-A, adenosine, and tubercidin, but not deoxyadenosine or deoxycytidine, was greatly reduced in intact L1210/ara-A cells, relative to L1210/C2 cells, and adenosine kinase activity in extracts of L1210/ara-A cells was negligible. Resistance to ara-A, and cross-resistance to tubercidin, methylmercaptopurine ribonucleoside, and cordycepin is attributed to loss of adenosine kinase activity.

Differential activity of vincristine and vinblastine against cultured cells.

Vincristine and vinblastine exhibit differential activity against tumors and normal tissues. In this work, a number of cultured cell lines were assayed for their sensitivity to the antiproliferative and cytotoxic effects of the two drugs following short-term (4 hr) or during continuous exposures. Differential activity was not seen when cells were subjected to continuous exposures. The concentrations of vincristine and vinblastine, respectively, that inhibited growth rates by 50% were: mouse leukemia L1210 cells, 4.4 and 4.0 nM; mouse lymphoma S49 cells, 5 and 3.5 nM; mouse neuroblastoma cells, 33 and 15 nM; HeLa cells, 1.4 and 2.6 nM; and human leukemia HL-60 cells, 4.1 and 5.3 nM. In contrast, differential toxicity was seen when cells were subjected to 4-hr exposures and transferred to drug-free medium: the 50% growth-inhibitory concentrations for vincristine and vinblastine, respectively, for inhibition (a) of proliferation of L1210 cells were 100 and 380 nM and of HL-60 cells were 23 and 900 nM and (b) of colony formation of L1210 cells were 6 and greater than 600 nM and of HeLa cells were 33 and 62 nM. Uptake and release of [3H]-vincristine and [3H]vinblastine were examined in L1210 cells under the conditions of growth experiments. Uptake of both drugs was dependent on the pH of culture media, and significantly greater amounts of [3H]vinblastine than of [3H]vincristine were associated with cells after 4-hr exposures to equal concentrations of either drug. When cells were transferred to drug-free medium after 4-hr exposures, vinblastine was released much more rapidly from cells than was vincristine, and by 0.5 hr after resuspension of cells, the amount of vincristine associated with the cells was greater than the amount of vinblastine and remained so for up to at least 6 hr.

Effect of lithium on the myelosuppressive and chemotherapeutic activities of vinblastine.

Serum concentrations of lithium after i.p. administration to normal mice declined with biphasic kinetics. Treatment of mice bearing ascitic L1210 leukemia with lithium resulted in a modest protection against the myelosuppressive effects of vinblastine. Lithium as a single agent was without effect on the survival times of mice bearing either the L1210 or P388 leukemia. Similarly, there was no evidence of significant antiproliferative or cytotoxic activity when cultured L1210 leukemia and murine neuroblastoma cells were exposed to lithium at levels comparable to those observed in the serum of lithium-treated mice. The therapeutic activity of vinblastine against the L1210 and P388 leukemias was not significantly altered by either simultaneous or subsequent administration of lithium. Lithium did not antagonize the antiproliferative or cytotoxic action of vinblastine against L1210 leukemia or murine neuroblastoma cells and was without effect in experiments with neuroblastoma cells that assessed vinblastine inhibition of a biological function dependent on formation of cytoplasmic microtubules (neurite formation induced by serum deprivation). The results obtained suggest that administration of lithium to reduce myelosuppression is not likely to counteract the tumoricidal activity of vinblastine.

Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cell lines.

Gemcitabine (2',2'-difluorodeoxycytidine) is a novel pyrimidine nucleoside drug with clinical efficacy in several common epithelial cancers. We have proposed that gemcitabine requires nucleoside transporter (NT) proteins to permeate the plasma membrane and to exhibit pharmacological activity. In humans, there are seven reported distinct NT activities varying in substrate specificity, sodium dependence, and sensitivity to inhibition by nitrobenzylthioinosine (NBMPR) and dipyridamole. To determine which NTs are required for gemcitabine-dependent growth inhibition, cultures from a panel of 12 cell lines with defined plasma membrane NT activities were incubated with different concentrations of gemcitabine. Cell proliferation was assessed by the sulforhodamine B assay and cell enumeration to identify the concentrations of gemcitabine that inhibited cell replication by 50% (IC50s). NT activity was a prerequisite for growth inhibition in vitro because: (a) the nucleoside transport-deficient cells were highly resistant to gemcitabine; and (b) treatment of cells that exhibited only equilibrative NT activity with NBMPR or dipyridamole increased resistance to gemcitabine by 39- to 1800-fold. These data suggested that the type of NT activities possessed by a cell may be an important determinant of its sensitivity to gemcitabine and that NT deficiency may confer significant gemcitabine resistance. We analyzed the uptake kinetics of [3H]gemcitabine by each of five human NT activities in cell lines that exhibited a single NT activity in isolation; transient transfection of the cDNAs encoding the human concentrative NT proteins (hCNT1 and hCNT2) was used to study the cit and cif activities, respectively. The efficiency of gemcitabine uptake varied markedly among the cell lines with single NTs: es approximately = cit > ei > cib >>> cif. The transportability of [3H]gemcitabine was demonstrated by reconstitution of the human es NT in proteoliposomes, confirming that gemcitabine permeation is a protein-mediated process.

Mechanisms of uptake and resistance to troxacitabine, a novel deoxycytidine nucleoside analogue, in human leukemic and solid tumor cell lines.

Troxacitabine (Troxatyl; BCH-4556; (-)-2'-deoxy-3'-oxacytidine), a deoxycytidine analogue with an unusual dioxolane structure and nonnatural L-configuration, has potent antitumor activity in animal models and is in clinical trials against human malignancies. The current work was undertaken to identify potential biochemical mechanisms of resistance to troxacitabine and to determine whether there are differences in resistance mechanisms between troxacitabine, gemcitabine, and cytarabine in human leukemic and solid tumor cell lines. The CCRF-CEM leukemia cell line was highly sensitive to the antiproliferative effects of troxacitabine, gemcitabine, and cytarabine with inhibition of proliferation by 50% observed at 160, 20, and 10 nM, respectively, whereas a deoxycytidine kinase (dCK)-deficient variant (CEM/dCK(-)) was resistant to all three drugs. In contrast, a nucleoside transport-deficient variant (CEM/ARAC8C) exhibited high levels of resistance to cytarabine (1150-fold) and gemcitabine (432-fold) but only minimal resistance to troxacitabine (7-fold). Analysis of troxacitabine transportability by the five molecularly characterized human nucleoside transporters [human equilibrative nucleoside transporters 1 and 2, human concentrative nucleoside transporter (hCNT) 1, hCNT2, and hCNT3] revealed that short- and long-term uptake of 10-30 microM [(3)H]troxacitabine was low and unaffected by the presence of either nucleoside transport inhibitors or high concentrations of nonradioactive troxacitabine. These results, which suggested that the major route of cellular uptake of troxacitabine was passive diffusion, demonstrated that deficiencies in nucleoside transport were unlikely to impart resistance to troxacitabine. A troxacitabine-resistant prostate cancer subline (DU145(R); 6300-fold) that exhibited reduced uptake of troxacitabine was cross-resistant to both gemcitabine (350-fold) and cytarabine (300-fold). dCK activity toward deoxycytidine in DU145(R) cell lysates was <20% of that in DU145 cell lysates, and no activity was detected toward troxacitabine. Sequence analysis of cDNAs encoding dCK revealed a mutation of a highly conserved amino acid (Trp(92)-->Leu) in DU145(R) dCK, providing a possible explanation for the reduced phosphorylation of troxacitabine in DU145(R) lysates. Reduced deamination of deoxycytidine was also observed in DU145(R) relative to DU145 cells, and this may have contributed to the overall resistance phenotype. These results, which demonstrated a different resistance profile for troxacitabine, gemcitabine, and cytarabine, suggest that troxacitabine may have an advantage over gemcitabine and cytarabine in human malignancies that lack or have low nucleoside transport activities.

Punitive childhood experiences reported by young adults over a 10-year period.

Using a standardized questionnaire, descriptions of disciplinary childhood experiences were obtained from 11,660 university undergraduates over a 10-year period. Results indicated that there were no systematic changes in reports of physical discipline experienced by young adults over the decade sampled, suggesting the prevalence of physical abuse among predominately middle-income families is not evidencing any significant change. Additionally, regardless of whether their early childhood antedated or succeeded the growth in public attention to physical abuse, most young adults who reported severely punitive or injurious disciplinary events failed to categorize those experiences as abusive. The implications of the results are discussed in the context of efforts to raise public awareness of abuse and efforts to reduce possible transgenerational patterns of maltreatment.

Internal fixation of a displaced tibial sesamoid fracture.

The authors present a surgical technique for the preservation and repair of an acutely fractured sesamoid using internal fixation of the sesamoid. A case report demonstrating the technique for the open reduction and internal fixation of a fractured tibial sesamoid is presented. The authors recommend this procedure as a viable alternative to surgical excision of the tibial sesamoid. The use of the procedure as an adjunct for the surgical treatment of recalcitrant traumatic sesamoiditis is also discussed.

Tricorrectional bunionectomy for surgical repair of juvenile hallux valgus.

The authors propose the use of the tricorrectional bunionectomy as an alternate correction of severe deformity in juvenile hallux valgus. In the past, hallux valgus surgery in juveniles has been avoided. A follow-up study of the tricorrectional bunionectomy as the surgical treatment for juvenile bunion deformity in seven patients is presented.

Tricorrectional bunionectomy for correction of high intermetatarsal angles.

The authors propose the use of the tricorrectional bunionectomy as a viable procedure for correction of high intermetatarsal angles. The tricorrectional bunionectomy was performed on 39 patients (51 feet). All patients had intermetatarsal angles of 16 degrees or greater. The average follow-up period was 12.8 months. The authors believe this procedure is an excellent alternative to more disabling base osteotomies.

Tricorrectional osteotomy for the correction of late-stage hallux limitus/rigidus.

The authors propose the use of the tricorrectional osteotomy for treatment of severe hallux limitus/rigidus as an alternative to joint-destructive procedures. A study of 19 patients with follow-up treatment ranges of 10 months to 6 years postoperatively was performed. Data were collected on preoperative and long-term postoperative x-rays, range of motion assessment, F-scan studies, and subjective patient questionnaires. High patient satisfaction along with increased range of motion, minimal complications, and an early return to activities make this an ideal procedure for grades II, III, and IV hallux limitus/rigidus.

Tricorrectional bunionectomy for hallux abducto valgus. A comprehensive outcome study.

A longitudinal outcome study of the tricorrectional bunionectomy with AO screw fixation for the correction of hallux abducto valgus was undertaken involving 84 consecutive patients (121 feet) with a mean age of 48.4 years who underwent the procedure over a 6-month period. Preoperative and postoperative measurements of forefoot angles were calculated, with the following mean results obtained: intermetatarsal angle of 14.46 degrees corrected to 5.72 degrees, hallux abductus angle of 26.38 degrees corrected to 11.65 degrees, proximal articular set angle of 19.85 degrees corrected to 6.06 degrees, and tibial sesamoid position of 4.75 corrected to 1.87. The average time to return to athletic shoes was 12.63 days. Postoperative complications were minor, with no delayed unions, malunions, hematomas, bone infections, or hallux varus. Long-term follow-up (mean, 21.33 months) using the American Orthopaedic Foot and Ankle Society's objective Hallux Metatarsophalangeal-Interphalangeal Scale revealed an overall mean score of 88.94 points out of a possible 100. An excellent mean result of 95 points out of a possible 100 on the subjective patient rating scale was also reported.

Adult sequelae of childhood abuse presenting as environmental illness.

Sixty-three patients with polysomatic complaints attributed to sensitivity to environmental chemicals had detailed clinical assessments and diagnostic psychologic evaluations. Objective medical parameters failed to substantiate their beliefs that multiple chemicals were the cause of their problems. A group of 64 patients with chronic medical conditions and defined psychologic disorders not attributed to chemical exposure served as controls. Approximately half the patients in each group underwent long-term psychotherapy, and in these patients, the prevalence of physical and sexual childhood abuse was significantly higher (P < .05) among the cohort of women who attributed their symptoms to environmental or chemically related illness. These data suggest that somatization may reflect sequelae of childhood abuse and may play an important role in the illness experienced by women who believe they are sensitive to environmental chemicals.

Acquisition of human concentrative nucleoside transporter 2 (hcnt2) activity by gene transfer confers sensitivity to fluoropyrimidine nucleosides in drug-resistant leukemia cells.

CEM-ARAC leukemia cells with resistance to cytarabine were shown to lack equilibrative transporter (hENT1) expression and activity. Stable transfer of hCNT2 cDNA into CEM-ARAC enabled Na(+)-dependent transport of purine and pyrimidine nucleoside analogs and provided a unique in vitro model for studying hCNT2. Analysis of [(3)H]uridine inhibitory activity by test substances in hCNT2 transfectant ARAC/D2 revealed structural requirements for interaction with hCNT2: 1) ribosyl and 2'-deoxyribosyl nucleosides were better inhibitors than 3'-deoxyribosyl, 2',3'-dideoxyribosyl or arabinosyl nucleosides; 2) uridine analogs with halogens at position 5 were better inhibitors than 5-methyluridine or thymidine; 3) 2-chloroadenosine was a better inhibitor than 2-chloro-2'-deoxyadenosine (cladribine); and 4) cytosine-containing nucleosides, 7-deazaadenosine and nucleobases were not inhibitors. Quantification of inhibitory capacity yielded K(i) values of 34-50 microM (5-halogenated uridine analogs, 2'-deoxyuridine), 82 microM (5-fluoro-2'-deoxyuridine), 197-246 microM (5-methyluridine < 5-bromo-2'-deoxyuridine < 5-iodo-2'-deoxyuridine), and 411 microM (5-fluoro-5'-deoxyuridine, capecitabine metabolite). Comparisons of hCNT2-mediated transport rates indicated halogenated uridine analogs were transported more rapidly than halogenated adenosine analogs, even though hCNT2 exhibited preference for physiologic purine nucleosides over uridine. Kinetics of hCNT2-mediated transport of 5-fluorouridine and uridine were similar (K(m) values, 43-46 microM). The impact of hCNT2-mediated transport on chemosensitivity was assessed by comparing antiproliferative activity of nucleoside analogs against hCNT2-containing cells with transport-defective, drug-resistant cells. Chemosensitivity was restored partially for cladribine, completely for 5-fluorouridine and 5-fluoro-2'-deoxyuridine, whereas there was little effect on chemosensitivity for fludarabine, 7-deazaadenosine, or cytarabine. These studies, which demonstrated hCNT2 uptake of halogenated uridine analogs, suggested that hCNT2 is an important determinant of cytotoxicity of this class of compounds in vivo.

Comparison of the effects on cultured L1210 leukemia cells of the ribosyl, 2'-deoxyribosyl, and xylosyl homologs of tubercidin and adenosine alone or in combination with 2'-deoxycoformycin.

The biologic effects of a series of sugar-substituted analogs of tubercidin were evaluated and compared with the effects of the homologous series of adenosine analogs in combination with 2'-deoxycoformycin. The greatest cytotoxicity against cultured mouse L1210 leukemia cells was exhibited by tubercidin and by 3'-deoxyadenosine or xylosyladenine in combination with 2'-deoxycoformycin. Somewhat less active were xylotubercidin and the combination of arabinosyladenine (araA) plus 2'-deoxycoformycin. The arabinosyl and 2'- and 3'-deoxyribosyl derivatives of tubercidin were relatively ineffective in their ability to inhibit proliferation of L1210 cells. The major biochemical effects of the most active agents were inhibition of RNA synthesis (3'-deoxyadenosine and xylosyladenine) and depletion of cellular ATP plus general inhibition of macromolecular synthesis (tubercidin). The in vitro activities of xylosyladenine and 3'-deoxyadenosine (in combination with 2'-deoxycoformycin) and xylotubercidin (as a single agent) were greater than or equivalent to that of araA (in combination with 2'-deoxycoformycin).