MyD88 and Src are differentially regulated in Kupffer cells of males and proestrus females following hypoxia.

Hypoxia produces sex dimorphic immune responses in males and proestrus females. Because Kupffer cells are the major source of proinflammatory cytokines, studies were conducted to discern IL-6 production in mouse Kupffer cells following hypoxia. Hypoxia enhances TLR4 expression in Kupffer cells irrespective of sex. However, MyD88 and Src expression in Kupffer cells decreased significantly after hypoxia in proestrus females, whereas Src protein expression and phosphorylation increased in males in concurrence with differences in IL-6 production. 17beta-estradiol administration elevated MyD88 and Src expression in males to levels in normoxic proestrus females. Administration of Src inhibitor in hypoxic males prevented increased IL-6 production. Thus, differential regulation of MyD88 and Src in males and females plays an important role in sex-specific immune response following hypoxia.

Mouse genetic background influences severity of immune responses following trauma-hemorrhage.

Studies have shown that following bacterial infection or endotoxin administration, immune functions are regulated differently in mice of different genetic background. Since the susceptibility to sepsis following trauma-hemorrhage is dependant on the severity of injury, it is important to determine whether genetic background of the animal influence immune functions after trauma-hemorrhage. The aim of our studies, therefore, was to assess differences in the immune functions in genetically different strains of age-matched C3H/HeN and C57BL/6 male mice following trauma-hemorrhage. The analysis for immune functions included: proliferation of splenocyte and bone-marrow cells, IL-2 and IFN-gamma release by splenocytes, and TNF-alpha and IL-10 release by splenic, peritoneal, liver (Kupffer cell), and bone-marrow macrophages. The results show significant differences in splenocyte and bone-marrow functions, and in the release of the mediators of immune function by immune competent cells: (a) between the two genetic strains, and (b) in each mouse strain following trauma-hemorrhage. Thus, genetic background appears to significantly influence the severity of immune responses in males following trauma-hemorrhage.

Transgenic prolactin-/- mice: effect of trauma-hemorrhage on splenocyte functions.

Prolactin (PRL) is involved in the regulation of immune functions under normal and pathological conditions. Trauma-hemorrhage (T-H) produces profound immunosuppression in male mice but not in proestrus female mice. Administration of PRL in males after T-H, however, restores immune functions. In this study, PRL(+/+) and transgenic (PRL(-/-)) male and female mice were used to assess immune suppression after T-H and to determine the reasons for the hormone's beneficial effect. In vitro lymphoproliferation assay with Nb2 cells showed complete absence of PRL in the circulation of the transgenic PRL(-/-) mice of both sexes, whereas very high levels of the hormone were detected in the wild-type PRL(+/+) mice of both sexes. Moreover, T-H resulted in the appearance of significant levels of the hormone in circulation, but only in PRL(+/+) mice. Splenocyte proliferation in male PRL(-/-) mice was significantly lower than in PRL(+/+) mice after T-H. Marginal differences between PRL(+/+) and PRL(-/-) mice were observed in the release of IL-2 and IFN-gamma by splenocytes, while the release of IL-10 was significantly higher in PRL(-/-) than in PRL(+/+) mice. A significant observation of our study is the release of a approximately 25-kDa protein in the concanavalin A-stimulated splenocytes of male PRL(+/+) and PRL(-/-) mice that was active in the in vitro lymphoproliferation assay with Nb2 cells. It is unlikely that this protein is PRL because it is also present in the splenocyte extracts of PRL(-/-) transgenic mice. Nonetheless, because control of lymphoid cell proliferation is considered one of the characteristics of the immune system, the local release of this protein may be significant in the differences observed in splenocyte cytokine release after T-H in wild-type as well as transgenic mice.

Immunoprotection in proestrus females following trauma-hemorrhage: the pivotal role of estrogen receptors.

Immune responses in proestrus females are not altered after trauma-hemorrhage, whereas they are markedly depressed in males. Elevated levels of female sex steroids appear to be responsible for maintaining immune responses but it remains unknown, whether estrogen per se is responsible. To study this, proestrus female C3H/HeN mice were subjected to laparotomy (i.e., soft tissue trauma) and hemorrhagic shock (35+/-5 mmHg for 90 min, then resuscitated) or sham operation and received the estrogen receptor antagonist EM-800 or vehicle during resuscitation. Two hours following trauma-hemorrhage, splenocyte proliferation, IL-2, IL-3, IFN-gamma release, and splenic macrophage IL-6 release was maintained in vehicle-treated females. In EM-800-treated females, however, these immune parameters were significantly depressed. Following trauma-hemorrhage, Kupffer cell TNF-alpha release and circulating TNF-alpha were increased only in EM-800-treated females. These findings indicate that the ability of proestrus females to maintain immune function following trauma-hemorrhage is estrogen-dependent and mediated via estrogen receptors.

Endocrine targets in experimental shock.

Modified resuscitation regimens and cytokine blockade/receptor antagonism after trauma have not been successful in decreasing the mortality rates from sepsis in trauma patients; therefore, an alternative approach using endocrine targets as modulators or inhibitors may be useful. Information regarding the influence of gender and hormones on immune and cardiovascular responses after nonthermal trauma-hemorrhagic shock is, on the one hand, considerable but, on the other hand, disappointingly incomplete. Trauma-hemorrhagic shock produces gender dimorphic immune and cardiovascular responses; men exhibit cardiovascular depression and are immunosuppressed, whereas proestrus women do not show cardiovascular or immunologic depression under those conditions. Furthermore, experimental studies have demonstrated the use of hormones, hormone antagonists, sex steroids, and receptor antagonists as salutary adjuncts, without any adverse effects on gastrointestinal, hepatic, and renal functions, for restoring the depressed immune and cardiovascular responses after trauma-hemorrhage. Thus, flutamide, dehydroepiandrosterone, metoclopramide, and 17beta-estradiol, which are readily availably clinically and do not produce any adverse hemodynamic effects, appear to be safe and novel agents/hormones for the treatment of immune and cardiovascular depression after severe blood loss in male and female trauma victims.

Androgen-mediated modulation of macrophage function after trauma-hemorrhage: central role of 5alpha-dihydrotestosterone.

Androgens have been implicated as the causative factor for the postinjury immune dysfunction in males; however, it remains unknown whether androgens directly affect macrophages. To study this, male mice were sham operated or subjected to trauma (i.e., midline laparotomy) and hemorrhagic shock (mean arterial pressure, 30 +/- 5 mmHg for 90 min and then resuscitated). The mice received the 5alpha-reductase inhibitor 4-hydroxyandrostenedione (4-OHA) before resuscitation. Plasma TNF-alpha, IL-6, and IL-10 levels were elevated after trauma-hemorrhage and normalized by 4-OHA. TNF-alpha and IL-6 production by splenic macrophages was decreased after injury, whereas Kupffer cell production of these mediators was enhanced. 4-OHA normalized cytokine production. Androgens suppressed cytokine production by splenic macrophages from hemorrhaged mice, whereas it enhanced TNF-alpha and IL-6 production by Kupffer cells. The addition of 4-OHA in vitro normalized cytokine production by cells treated with testosterone, but it had no effect on dihydrotestosterone-treated cells. These results indicate that androgens directly affect macrophage function in males after trauma and hemorrhagic shock and that the intracellular conversion of testosterone to dihydrotestosterone is of particular importance in mediating the androgen-induced effects.

Mechanism for normal splenic T lymphocyte functions in proestrus females after trauma: enhanced local synthesis of 17beta-estradiol.

Trauma-hemorrhage and resuscitation (TH) produces profound immunodepression and enhances susceptibility to sepsis in males but not in proestrus females, suggesting gender dimorphism in the immune responses. However, the mechanism responsible for the maintenance of immune functions in proestrus females after TH is unclear. Splenic T lymphocytes express receptors for estrogen (ER), contain enzymes involved in estrogen metabolism, and are the major source of cytokine production; the metabolism of 17beta-estradiol was assessed in the splenic T lymphocytes of proestrus and ovariectomized mice by using appropriate substrates after TH. Analysis for aromatase and 17beta-hydroxysteroid dehydrogenases indicated increased 17beta-estradiol synthesis and low conversion into estrone in T lymphocytes of proestrus but not of ovariectomized mice. The effect of 17beta-estradiol on T lymphocyte cytokine release was reliant on ER expressions. This was apparent in the differences of ER expression, especially that of ER-beta, and an association between increased 17beta-estradiol synthesis and sustained release of IL-2 and IL-6 in T lymphocytes of proestrus females after TH. Because 17beta-estradiol is able to regulate cytokine genes, and the splenic T lymphocyte cytokine releases is altered after TH, continued synthesis of 17beta-estradiol in proestrus females appears to be responsible for the maintenance of T lymphocyte cytokine release associated with the protection of immune functions after TH.

Gender dimorphism in immune responses following trauma and hemorrhage.

Previous studies have shown marked immunosuppression in males after trauma and hemorrhage. By contrast, immunosuppression was not evident in proestrous female animals. Further support for these findings came from studies demonstrating the immunosuppressive effects of male sex hormones and immunoenhancing effects of female sex hormones. In this regard, administration of 17beta-estradiol or prolactin to male animals after trauma and hemorrhage significantly improved immune function. Furthermore, castration or androgen receptor blockade with flutamide after trauma and hemorrhage in male mice showed similar beneficial effects. Thus, regulation of sex hormone synthesis or their receptor activity appears to be a useful therapeutic approach for patients to cope with the deleterious sequelae of severe trauma and hemorrhage.

Decreased 5alpha-dihydrotestosterone catabolism suppresses T lymphocyte functions in males after trauma-hemorrhage.

Trauma-hemorrhage produces profound immunosuppression in males but not in proestrus females. Prior castration or flutamide treatment of males following trauma-hemorrhage prevents immunosuppression, implicating 5alpha-dihydrotestosterone for the immunosuppressive effects. 5alpha-dihydrotestosterone, a high-affinity androgen receptor-binding steroid, is synthesized in tissues as needed and seldom accumulates. The presence of steroidogenic enzymes in T lymphocytes suggests both synthesis and catabolism of 5alpha-dihydrotestosterone. We hypothesized, therefore, that the basis for high 5alpha-dihydrotestosterone activity in T lymphocytes of males following trauma-hemorrhage is due to decreased catabolism. Accordingly, catabolism of 5alpha-dihydrotestosterone was assessed in splenic T lymphocytes by examining the activity and expression of enzymes involved. Analysis showed increased synthesis and decreased catabolism of 5alpha-dihydrotestosterone in intact male T lymphocytes following trauma-hemorrhage. In contrast, reduced 5alpha-reductase activity and increased expression of 17beta-hydroxysteroid dehydrogenase oxidative isomers suggest inactivation of 5alpha-dihydrotestosterone in precastrated males. Thus our study suggests increased synthesis and decreased catabolism of 5alpha-dihydrotestosterone as a reason for loss of T lymphocyte functions in intact males following trauma-hemorrhage, as evidenced by decreased release of interleukin-2 and -6.

Divergent immune responses in male and female mice after trauma-hemorrhage: dimorphic alterations in T lymphocyte steroidogenic enzyme activities.

Immune responses are suppressed in males, but not in proestrous females, after trauma-hemorrhage. Testosterone and 17beta-estradiol appear to be responsible for divergent immune effects. There is considerable evidence to suggest sex steroid hormone involvement in immune functions. As formation of active steroid depends on the activity of androgen- and estrogen-synthesizing enzymes, expression and activity of 5alpha-reductase, aromatase, and 3beta- and 17beta- hydroxysteroid dehydrogenases were determined in spleen and T lymphocytes of male and proestrous female mice after trauma-hemorrhage. All of the enzymes were present in spleen, specifically in T lymphocytes. 5alpha-Reductase expression and activity increased in male T lymphocytes, whereas aromatase activity, but not expression, increased in female T lymphocytes. Increased 5alpha-reductase activity in male T lymphocytes is immunosuppressive because of increased 5alpha-dihydrotestosterone synthesis, whereas in females increased aromatase activity triggering 17beta-estradiol synthesis is immunoprotective. This study also demonstrates the importance of 17beta-hydroxysteroid dehydrogenase oxidative and reductive functions. The immunoprotection of proestrous females is associated with enhanced reductase function of the enzyme. In males, decreased expression of oxidative isomer type IV, which impairs catabolism of 5alpha-dihydrotestosterone, probably augments immunosuppression. This study provides evidence for the involvement of intracrine sex steroid synthesis in gender dimorphic immune responses after trauma-hemorrhage.

Androgen and estrogen receptors in splenic T lymphocytes: effects of flutamide and trauma-hemorrhage.

The endogenous sex steroids, testosterone and beta-estradiol, play a major role in inflammatory processes. They regulate several cytokine genes by interaction with their intracellular receptors that are, essentially, transcription factors. Because T-lymphocyte functions are altered following trauma-hemorrhage in male mice, we investigated whether (i) receptors for androgen (AR) and estrogen (ER) are present in splenic T lymphocytes, (ii) receptor expressions are altered following trauma-hemorrhage, and (iii) pretreatment of male mice with the AR antagonist, flutamide, alters receptor expressions and IL-6 release. Analysis of nuclear extracts indicated the presence of AR and ER in splenic T lymphocytes. No difference in receptor expressions between males and females or following trauma-hemorrhage was observed. Pretreatment of males with flutamide, however, led to increased ER expression in T lymphocytes of sham and trauma-hemorrhaged animals. This suggested that flutamide is capable of inducing the expression of another receptor belonging to a different gonadal steroid. Because response elements for AR and ER are present in the promoter region of the IL-6 gene, release of IL-6 and expression of signal transducer and activator of transcription 3 (STAT3) were analyzed as functional parameters in splenic T lymphocytes. Trauma-hemorrhage decreased IL-6 release by T lymphocytes and the release was restored to sham levels with flutamide pre-treatment. Similarly, STAT3 expression was decreased in T lymphocytes following trauma-hemorrhage and the expression was restored by flutamide pre-treatment. These data collectively demonstrate the importance of gonadal steroids in the regulation of splenic T-lymphocyte functions.

The aromatase inhibitor, 4-hydroxyandrostenedione, restores immune responses following trauma-hemorrhage in males and decreases mortality from subsequent sepsis.

Studies have shown that immune responses are depressed in male mice, but not in proestrus females after trauma-hemorrhage (TH), resulting in increased mortality from subsequent sepsis in male mice compared with female mice. These gender-specific alterations in immune function are believed to be due to differences in sex steroid levels. Aromatase is a key enzyme in the sex steroid biosynthesis. Although earlier studies have shown that aromatase inhibitors prevent thymic atrophy in aged male rats, it remains unknown whether the use of 4-hydroxy-androstenedione (4-OHA) after TH in male mice has any salutary effects on the depressed immune responses. Male C3H/HeN mice were sham operated or subjected to trauma (i.e., midline laparotomy) and hemorrhagic shock (30+/-5 mmHg for 90 min) followed by adequate fluid resuscitation. 4-OHA (5 mg/kg) or vehicle was administrated s.c. just before resuscitation. At 2 h after resuscitation, the mice were killed, and spleens were harvested. Splenocyte proliferation, interleukin (IL-2), interferon (IFN-gamma), and IL-10 release and expression of androgen (AR) and estrogen receptors (ER)-alpha and -beta by immunoblot and reverse transcription-polymerase chain reaction (RT-PCR) were assessed. In another group, sepsis was induced by cecal ligation and puncture (CLP) 3 days after resuscitation, and survival was measured over a period of 10 days. A significant decrease in splenocyte proliferation, IL-2, and IFN-gamma release and increased release of IL-10 were observed in vehicle-treated mice. Animals treated with 4-OHA showed increased splenocyte proliferation, IL-2, and IFN-gamma release, and decreased IL-10 release. Immunoblot analysis showed decreased expression of the cytosolic AR, but no significant difference in the cytosolic and nuclear ER-alpha and -beta expression was observed in the vehicle-treated group after TH. In addition, AR and ER-beta mRNA expression was increased, whereas ER-alpha expression decreased in the vehicle-treated group after TH. ER-alpha expression decreased and ER-beta expression increased in the nucleus of 4-OHA treated mice as determined by immunoblot. There was no difference in the cytosolic AR expression in the 4-OHA-treated group after TH. AR and ER-beta mRNA expression was unaffected, whereas ER-alpha expression increased under such conditions. In additional groups, the increased mortality rate after TH and subsequent sepsis was significantly reduced by 4-OHA treatment. Thus, 4-OHA seems to be a novel and useful adjunct for restoring the depressed immune functions in males after TH and for decreasing mortality rates from subsequent sepsis.

Proteasome participates in the alteration of signal transduction in T and B lymphocytes following trauma-hemorrhage.

Proteasomes are essential components of the cellular protein degradation machinery. They are nonlysosomal and their participation is critical for (1) the removal of short lived proteins involved in metabolic regulation and cell proliferation, (2) the control of the activities of regulators involved in gene transcription, such as nuclear factor-kappa B (NF-kappa B) and signal transducer and activator of transcription (STAT1), and (3) processing of antigenic peptides for MHC class I presentation. Trauma-hemorrhage induces profound immunosuppression which is characterized by reduced splenocyte proliferation, interleukin (IL)-2 and interferon (IFN)-gamma productive capacity, increased activation of transcription factors NF-kappa B and STAT1 in splenic T lymphocytes, reduced macrophage antigen presentation capacity and inordinate release of proinflammatory cytokines, such as IL-6 and tumor necrosis factor-alpha. Furthermore, it appears that the activity of several regulatory proteins involved in immune function is altered by trauma-hemorrhage. Since proteasomes are involved in regulation and removal of regulatory proteins, we hypothesized that trauma-hemorrhage alters proteasomal activity in splenic lymphocytes. The data showed that activities of 26s proteasome from CD3+CD4+ and CD3+CD8+ splenic T lymphocytes were enhanced following trauma-hemorrhage which was associated with increased expression of NF-kappa B and STAT1. On the other hand, trauma-hemorrhage attenuated the activity of 26s proteasome from splenic B lymphocytes which was restored upon IFN-gamma stimulation and correlated with increased expression of NF-kappa B. These studies indicate a potential role for proteasomes in the regulation of signal transduction in splenic T and B lymphocytes following trauma-hemorrhage, and also suggest them as potential therapeutic targets for attenuation of immune suppression associated with this form of injury.

Thermal injury alters macrophage responses to prostaglandin E2: contribution to the enhancement of inducible nitric oxide synthase activity.

Prostaglandin E2 (PGE2) and macrophage (Mphi)-derived reactive nitrogen intermediates (RNI) have been implicated in T cell dysfunction after thermal injury. Normally, Mphi inducible nitric oxide synthase (iNOS) activity can be regulated by PGE2, however, it is unknown whether PGE2 modulates Mphi iNOS activity after thermal injury. Splenic Mphi isolated from mice 7 days after thermal injury produced higher levels of RNI than Mphi from sham mice when stimulated with lipopolysaccharide (LPS) or interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) in combination. PGE2, when added concurrently with LPS, suppressed RNI production by Mphi from sham mice, whereas Mphi from injured mice were unaffected. When Mphi were pretreated with PGE2 before LPS, RNI production was suppressed in both populations. RNI production in response to IFN-gamma or IFN-gamma and TNF-alpha in combination was enhanced by PGE2 in both populations, however, the effect was markedly greater in Mphi from injured mice. The PGE2-mediated changes in RNI production were paralleled by similar changes in iNOS protein expression, suggesting that the effect of PGE2 was at the level of enzyme expression rather than activity. Dibutryl cAMP induced similar effects as PGE2, suggesting the response to PGE2 after thermal injury is independent of potential changes in PGE2-induced adenylate cyclase activity and is cAMP-mediated. The results indicate that Mphi from burned mice display an altered sensitivity to PGE2, resulting in enhanced iNOS activity. Thus, PGE2, which is elevated after thermal injury and can directly suppress T cell function, may also contribute to immune dysfunction through the enhancement of Mphi iNOS activity.

Trauma-hemorrhage activates signal transduction pathways in mouse splenic T cells.

Severe impairment in the functions of immune-competent cells has been observed following trauma and hemorrhage. Inappropriate release of cytokines during trauma and hemorrhagic shock disrupt T lymphocyte functions and enable cells to activate genes whose products are detrimental for maintaining a much-needed humoral and cell-mediated immunity. The intracellular events for gene activation are mediated by cytoplasmic transcription factors present as nascent (signal transducer and activator of transcription 1 (STAT 1)) or as a complex (nuclear factor kappaB (NF-kappaB)). Receptor-initiated phosphorylation activates these transcription factors prior to their nuclear translocation and binding to cognate DNA sequences. Because T cell functions are critical to efficient functioning of the immune system, we investigated whether expression of transcription factors, STAT1 and NF-kappaB, is perturbed in splenic T cells following trauma and hemorrhage. To study this, enriched T cells harvested from spleens (pooled from three or four mice per group) of sham, trauma (consisting of midline laparotomy), sham+trauma, hemorrhage (blood pressure maintained at approximately 30 mmHg for 90 min followed by adequate fluid resuscitation), and trauma+hemorrhage groups at 16-18 h after surgical procedure were probed for signal expressions in the presence and absence of interferon-gamma using electrophoretic mobility shift and Western immunoblot assay procedures. Hemorrhage with or without trauma induced activation of Janus kinase 1, STAT1, and NF-kappaB in T cells. Stimulation of T cells with interferon-gamma led to activation of all these signals in all groups including experimental controls. STAT1 activation was accompanied by Janus kinase 1 phosphorylation, whereas NF-kappaB activation was mediated by phosphorylation and rapid degradation of IkappaBalpha. These studies demonstrate that hemorrhagic shock, with or without laparotomy, is sufficient to induce activation of transcription factors in splenic T cells. Thus, attempts to prevent the activation of transcription factors following hemorrhage by pharmacologic means might be helpful for maintaining cell-mediated immunity under these conditions.

Phase I and pharmacokinetics studies of prochlorperazine 2-h i.v. infusion as a doxorubicin-efflux blocker.

In an earlier phase I study, we reported that the maximal tolerated dose (MTD) of prochlorperazine (PCZ) given as a 15-min i.v. infusion was 75 mg/m2. The highest peak plasma PCZ concentration achieved was 1100 ng/ml. The present study was conducted to determine if PCZ levels high enough to block doxorubicin (DOX) efflux in vitro could be achieved and sustained in vivo by increasing the duration of i.v. infusion from 15 min to 2 h. The treatment schedule consisted of i.v. prehydration with at least 500 ml normal saline (NS) and administration of a fixed standard dose of 60 mg/m2 DOX as an i.v. bolus over 15 min followed by i.v. doses of 75, 105, 135, or 180 mg/m2 PCZ in 250 ml NS over 2 h. The hematologic toxicities attributable to DOX were as expected and independent of the PCZ dose. Toxicities attributable to PCZ were sedation, dryness of mouth, anxiety, akathisia, hypotension, cramps, and confusion. The MTD of PCZ was 180 mg/m2. Large interpatient variation in peak PCZ plasma levels (91-3215 ng/ml) was seen, with the plasma half-life (t1/2 alpha) being approximately 57 min in patients given 135-180 mg/m2 PCZ. The volume of distribution (Vd), total clearance (ClT), and area under the curve (AUC) were 350.1 +/- 183.8 1/m2, 260.7 +/- 142.7 l m2 h-1 and 1539 +/- 922 ng ml h-1, respectively, in patients given 180 mg/m2 PCZ and the respective values for patients receiving 135 mg/m2 were 48.9 +/- 23.76 l/m2, 33.2 +/- 2.62 l m2 h-1, and 4117 +/- 302 ng ml h-1. High PCZ plasma levels (> 600 ng/ml) were sustained in all patients treated with 135 mg/m2 PCZ for up to 24 h. DOX plasma elimination was biphasic at 135 and 180 mg/m2 PCZ, and a > 10-ng/ml DOX plasma level was maintained for 24 h. Partial responses were seen in three of six patients with malignant mesothelioma, in two of ten patients with non-small-cell lung carcinoma, and in the single patient with hepatoma. Our data show that PCZ can be safely given as a 2-h infusion at 135 mg/m2 with clinically manageable toxicities. The antitumor activity of the combination of DOX and PCZ needs to be confirmed in phase II trials.

Doxorubicin-induced DNA breaks, topoisomerase II activity and gene expression in human melanoma cells.

We have analyzed five human melanoma cell lines, displaying variable doxorubicin resistance (1- to 6-fold), for drug-induced DNA breaks, topoisomerase II activity and mRNA expression. Enhanced drug efflux was not the reason for doxorubicin resistance of these tumor cells although they overexpressed the transmembrane 170 kDa P-glycoprotein. Doxorubicin-induced DNA lesions (2-fold) and topoisomerase II activity (7-fold) were higher in HM-1 and G361 cells than in the less doxorubicin-sensitive NH and FCCM-9 cells. Topoisomerase II mRNA expression was also 2-fold higher in HM-1 and G361 cells. Doxorubicin-induced DNA breaks and topoisomerase II activity inversely correlated with the degree of doxorubicin sensitivity. Southern blot analysis showed variation in the hybridization pattern of topoisomerase II gene in doxorubicin-resistant cells when compared to sensitive cells. This study portrays the low doxorubicin sensitivity of NH and FCCM-9 cells as "atypical" and emphasizes the importance of DNA damage and topoisomerase II activity in cellular low doxorubicin resistance.

Prochlorperazine as a doxorubicin-efflux blocker: phase I clinical and pharmacokinetics studies.

Doxorubicin (DOX) efflux in drug-resistant cells is blocked by phenothiazines such as trifluoperazine (TFP) and prochlorperazine (PCZ) in vitro. The present phase I study was conducted in 13 patients with advanced, incurable, nonhematologic tumors to determine whether PCZ plasma levels high enough to block DOX efflux could be achieved in vivo. The treatment schedule consisted of prehydration and i.v. administration of 15, 30, 50, and 75 mg/m2 PCZ followed by a standard dose of 60 mg/m2 DOX. The hematologic toxicities attributable to DOX were as expected and independent of the PCZ dose used. Toxicities attributable to PCZ were sedation, dryness of the mouth, cramps, chills, and restlessness. The maximal tolerated dose (MTD) of PCZ in this schedule was 75 mg/m2. Pharmacokinetic analysis indicated a large interpatient variation in peak plasma PCZ levels that ranged from 95 to 1100 ng/ml. The three plasma half-lives of PCZ were: t1/2 alpha (+/- SE), 20.9 +/- 5.3 min; t1/2 beta, 1.8 +/- 0.3 h; and t1/2 gamma, 21.9 +/- 5.3 h. The volume of distribution (Vd), total clearance (ClT), and area under the curve (AUC) for PCZ were 2254 +/- 886 l/m2, 60.2 +/- 13.5 l m-2 h-1, and 1624 +/- 686 ng ml-1 h, respectively. DOX retention in tumor cells retrieved from patients during the course of therapy indicated the appearance of cells with enhanced DOX retention. The combination of DOX and high-dose i.v. PCZ appeared to be safe, well tolerated, and active in non-small-cell lung carcinoma.

A phase I study of 4'-0-tetrahydropyranyladriamycin. Clinical pharmacology and pharmacokinetics.

A Phase I study of intravenous (IV) bolus 4'-0-tetrahydropyranyladriamycin (Pirarubicin) was done in 55 patients in good performance status with refractory tumors. Twenty-six had minimal prior therapy (good risk), 23 had extensive prior therapy (poor risk), and six had renal and/or hepatic dysfunction. A total of 167 courses at doses of 15 to 70 mg/m2 were evaluable. Maximum tolerated dose in good-risk patients was 70 mg/m2, and in poor-risk patients, 60 mg/m2. The dose-limiting toxic effect was transient noncumulative granulocytopenia. Granulocyte nadir was on day 14 (range, 4-22). Less frequent toxic effects included thrombocytopenia, anemia, nausea, mild alopecia, phlebitis, and mucositis. Myelosuppression was more in patients with hepatic dysfunction. Pharmacokinetic analyses in 21 patients revealed Pirarubicin plasma T 1/2 alpha (+/- SE) of 2.5 +/- 0.85 minutes, T beta 1/2 of 25.6 +/- 6.5 minutes, and T 1/2 gamma of 23.6 +/- 7.6 hours. The area under the curve was 537 +/- 149 ng/ml x hours, volume of distribution (Vd) 3504 +/- 644 l/m2, and total clearance (ClT) was 204 + 39.3 l/hour/m2. Adriamycinol, doxorubicin, adriamycinone, and tetrahydropyranyladriamycinol were the metabolites detected in plasma and the amount of doxorubicin was less than or equal to 10% of the total metabolites. Urinary excretion of Pirarubicin in the first 24 hours was less than or equal to 10%. Activity was noted in mesothelioma, leiomyosarcoma, and basal cell carcinoma. The recommended starting dose for Phase II trials is 60 mg/m2 IV bolus every 3 weeks.

Sequential 1H NMR assignments and secondary structure of aponeocarzinostatin in solution.

Sequential assignments and secondary structural analysis have been accomplished for the 113-residue apoprotein of the antitumor drug neocarzinostatin (NCS) from Streptomyces carzinostaticus. A total of 98% of the main-chain and 77% of the side-chain resonances have been sequence specifically assigned by use of information from coherence transfer experiments and by sequential and interstrand NOEs. Because of the complexity of the NCS spectrum, several sequential assignment strategies were employed to complete the analysis. Apo-NCS consists of three antiparallel beta-sheeted domains by NMR analysis. There is an extensive four-strand antiparallel beta-sheet, and two two-stranded domains. One of the two-strand domains is contiguous, S72-N87, with chain reversal occurring through the region L77-R82. The other two-stranded domain has the section G16-A24 antiparallel with respect to the region S62-R70. This secondary structure is consistent with the crystal structure of holo-NCS at 2.8-A resolution.