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The genetically related ACI and Copenhagen (COP) rat strains display diametrically opposed susceptibilities to mammary cancer development when treated chronically with 17beta-estradiol (E2). Here, we compare the actions of E2 on cell proliferation and lobuloalveolar development in the mammary glands of female ACI and COP rats. After 12 wk of E2 treatment, the mammary glands of ACI rats exhibited a significantly greater proliferative response to E2, compared with COP rats, as evidenced by quantification of S phase fraction and development of lobuloalveolar hyperplasia. Focal regions of atypical epithelial hyperplasia were observed in ACI, but not COP, rats. These strain differences were not because of differences in circulating E2, progesterone or, prolactin. Two-thirds of the induced mammary cancers in ACI rats exhibited aneuploidy. The E2-induced mammary cancers regressed when hormone treatment was discontinued, indicating that they were estrogen-dependent. Progesterone receptor was expressed by the great majority of epithelial cells within the E2-induced atypical hyperplastic foci and the mammary carcinomas, suggesting a link between these lesions. These data demonstrate a correlation between E2 action in the induction of mammary cell proliferation and atypical epithelial hyperplasia and genetically conferred susceptibility to E2-induced mammary cancers.

We have demonstrated that a 40% restriction of dietary energy consumption virtually abolishes the development of prolactin (PRL)-producing pituitary tumors in Fischer 344 (F344) rats treated chronically with estrogen, apparently by inhibiting the ability of estrogen to enhance survival within a rapidly proliferating lactotroph population. The purpose of the study reported here was to determine whether energy restriction exerts a similar antitumorigenic action in another rat strain, August x Copenhagen-Irish (ACI), in which PRL-producing pituitary tumors develop in response to estrogen treatment. Ovariectomized female ACI rats were either allowed to consume a control diet ad libitum or were fed a modified diet that restricted energy consumption by 40% relative to the amount of energy consumed by animals fed the control diet. We also examined the ability of 17beta-estradiol (E2) administered for 20 wk via subcutaneous Silastic implants to induce development of PRL-producing pituitary tumors. Treatment with E2 increased pituitary weight as well as the pituitary weight-to-body weight ratio and induced gross hyperprolactinemia to the same extent in ACI rats fed either the control or the energy-restricted diet. Moreover, dietary energy restriction did not affect the ability of E2 to induce pituitary cell proliferation or inhibit apoptosis, as evidenced by quantification of two surrogate markers. These data provide compelling evidence that a 40% restriction of energy consumption does not inhibit the ability of E2 to induce pituitary tumor development in the ACI rat. In conjunction with our published studies of the F344 rat strain, the data presented herein indicate that the inhibitory effects of dietary energy restriction on estrogen-induced pituitary tumor development are rat-strain specific and suggest that sensitivity to specific antitumorigenic actions of energy restriction is strongly affected by genetic background., (Copyright 1999 Wiley-Liss, Inc.)

IkappaBalpha is an inherently unstable protein which binds to and retains the ubiquitous transcription factor NFkappaB in the cytoplasm of resting cells. A continuous low level translocation of NFkappaB to the nucleus, secondary to the basal turnover of IkappaBalpha, is hypothesized to be necessary for cellular maturation, survival and, potentially, transformation. In response to cellular stimulation by inflammatory cytokines or mitogens, IkappaBalpha is rapidly degraded allowing larger pools of NFkappaB to translocate to the nucleus. Phosphorylation of IkappaBalpha at serine 32 (S32) and serine 36 (S36) is necessary for this stimuli-induced degradation. IKKalpha/beta kinases and p90(rsk1)are involved in stimuli-induced targeting of one or both of these IkappaBalpha sites. Whether other kinases phosphorylate S32 and S36 directly, and if so, what function they serve in NFkappaB activation remains unknown. Here we present evidence of a direct phosphorylation of IkappaBalpha at both S32 and S36 by purified or immunoprecipitated protein kinase CKII (PK-CKII) and a specific in vivo association between IkappaBalpha and PK-CKII. This PK-CKII-specific kinase activity is not found within the IKKalpha/beta-containing signalsome complex and is biochemically distinct from that of the IKKalpha/beta kinases. The identification of an additional N-terminal IkappaBalpha kinase which is constitutively active and not significantly inducible raises numerous possibilities as to its role in cellular function., (Copyright 1999 Academic Press.)

Estrogens stimulate cell proliferation in a variety of tissues and are widely believed to be contributing factors in the etiology of certain cancer types in humans. The molecular mechanisms through which estrogens regulate cell proliferation are currently unknown. Estrogens stimulate proliferation of the PRL-producing lactotroph of the rat anterior pituitary gland and induce development of PRL-producing pituitary tumors in several inbred rat strains. Therefore, the lactotroph provides a well defined model for identifying the mechanisms through which estrogens regulate cell proliferation and/or survival. Data from our laboratory and others indicate that the relative sensitivity to the pituitary growth-promoting actions of estrogens is highly strain specific. This allows genetics-based approaches to be used to address the molecular mechanisms through which estrogens stimulate lactotroph proliferation and induce pituitary tumor development. In the present study we have examined the ability of diethylstilbestrol (DES) to induce pituitary growth in the genetically related AxC-Irish (ACI) and Copenhagen (COP) strains and their derived F1, F2, and backcross progeny. The data presented herein indicate that the anterior pituitary gland of the ACI strain displays approximately a 2-fold greater growth response to administered DES than does the pituitary gland of the COP strain. The average pituitary weight in male ACI rats was increased from 9.2 +/- 0.2 mg (mean +/- SD in untreated rats to 63.7 +/- 12.6 mg in rats treated with DES for 12 weeks, whereas in male COP rats, DES increased pituitary weight from 12.7 +/- 0.9 to 38.1 +/- 8.2 mg. The ACI phenotype was inherited in the F1, F2, and backcross progeny of an ACI x COP intercross as a dominant genetic trait, and the approximately 30 mg of additional pituitary growth displayed by the DES-treated ACI rat, relative to that of the treated COP rat, appeared to result from the actions of a single locus. Moreover, in F1 progeny from an ACI x Brown Norway intercross, the ACI phenotype was inherited as a dominant or incompletely dominant genetic trait. These data, when compared with findings of previous studies using the Fischer 344 rat strain, provide the first indication that distinct genetic pathways contribute to regulation of estrogen-induced pituitary growth and induction of PRL-producing pituitary tumors in the ACI and F344 rat strains.

The goal of the present study was to test the hypothesis that a second, more distal, promoter exists upstream from the rat prolactin (PRL) gene (rPrl) that is homologous to that identified upstream from the human prolactin gene (hPrl). The nucleotide sequence of the rat genome extending from 7.4 to 2.5 kb upstream from the proximal rPrl promoter was determined, revealing significant sequence homology to the hPrl distal promoter and its 5'-flanking domain. Using reverse transcription-polymerase chain reaction, novel rPrl transcripts that originate upstream from the proximal rPrl promoter were detected in rat uterus, spleen, pons medulla, anterior pituitary and the GH4C1 pituitary tumor cell line. Further characterization of these novel 5'-extended rPrl transcripts from GH4C1 cells indicated that they were full length, polyadenylated and properly spliced. However, data from primer extension (5'-RACE) experiments strongly suggested that the 5'-extended rPrl transcripts originate, not at the distal promoter-like motif, but at scattered sites located 60-153 bp upstream from the proximal promoter. Therefore, it appears improbable that the rat sequences homologous to the hPrl distal promoter comprise a functional promoter.

Reduction in energy consumption is known to inhibit development of a variety of spontaneous, carcinogen-induced, and hormone-dependent cancers, but the mechanism or mechanisms by which this occurs remain unknown. We hypothesize that energy consumption may modulate development of estrogen-dependent neoplasms by altering the manner in which target cells respond to estrogens. To test this hypothesis, ovariectomized female Fischer 344 rats were fed diets that allowed consumption of different amounts of energy, and the ability of 17beta-estradiol (E2), administered for 10 wk from subcutaneous Silastic implants, to promote development of prolactin-producing pituitary tumors was examined. A 40% restriction of energy consumption virtually abolished the ability of E2 to promote development of pituitary tumors and associated hyperprolactinemia. A 25% restriction of energy consumption appeared to slightly inhibit E2-induced pituitary growth and hyperprolactinemia, but the observed degree of inhibition was not statistically significant. Interestingly, dietary energy restriction did not inhibit induction by E2 of pituitary cell proliferation and lactotroph hyperplasia. Furthermore, E2 treatment inhibited expression of testosterone-repressed prostate message-2 mRNA, a cellular marker of apoptosis, and this inhibitory effect of E2 was blocked by 40% energy restriction. These data suggest that dietary energy restriction virtually abolished E2-induced development of prolactin-producing pituitary tumors, not by blocking the ability of E2 to induce cell proliferation but rather by blocking the ability of E2 to enhance cell survival. This study and the accompanying paper provide the first indication that dietary energy consumption may modulate estrogen action at the level of the target cell.

Our laboratory is examining the hypothesis that diet may modulate the ability of estrogens to regulate cell proliferation and survival, either of which could affect development of neoplasms in estrogen-responsive tissues. In this study, we examined whether the amount of energy and protein consumed in the diet modulates the ability of the synthetic estrogen diethylstilbestrol (DES) to induce development of prolactin-producing pituitary tumors in two strains of rat, Fischer 344 (F344) and Holtzman, that differ in their propensity to develop pituitary tumors when treated with estrogens. Male F344 rats treated with DES for 8 wk developed pituitary tumors (defined as grossly enlarged pituitary masses that displayed diffuse lactotroph hyperplasia but lacked adenomatous foci). In contrast, male Holtzman rats displayed only a modest increase in pituitary weight in response to DES. Energy consumption but not protein consumption modulated DES-induced pituitary tumorigenesis in the male F344 rat. Relative to that observed in untreated animals, pituitary weights in F344 rats treated with DES increased 11.2- and 9.2-fold in animals fed either the control diet or an equicaloric high-protein diet, respectively, but only 3.5-fold in animals fed an energy-restricted diet. In contrast, neither the amount of energy nor protein consumed in the diet affected the modest pituitary growth response of male Holtzman rats to administered DES. Energy restriction had no apparent effect on pituitary cell proliferation, either basal or DES stimulated, in these rat strains. We concluded that dietary energy restriction inhibits the ability of administered DES to induce pituitary tumor development in the F344 rat by acting at a step after induction of pituitary cell proliferation.

The Copenhagen (COP) rat is unique among inbred rat strains in its high degree of resistance to spontaneously arising and induced mammary cancers. Hyperprolactinemia resulting from tumors of the anterior pituitary gland has been suggested to be the causative factor in the etiology of estrogen-induced mammary cancer in rats. Therefore, we have examined the ability of administered estrogens to induce development of PRL-producing pituitary tumors and mammary carcinomas in COP rats. Diethylstilbestrol (DES), administered to male COP rats for 12 weeks, beginning when the animals were 9 weeks of age, induced development of PRL-producing pituitary tumors, defined as grossly enlarged pituitary masses displaying lactotroph hyperplasia and associated hyperprolactinemia. When treated with 17beta-estradiol (E2), female COP rats developed pituitary tumors and hyperprolactinemia, but displayed a high degree of resistance to development of mammary carcinomas. These data indicate that E2-induced hyperprolactinemia is insufficient to induce development of mammary carcinomas in the female COP rat.

Previous investigations suggested potential breast cancer-preventive properties of dietary fiber from cabbage. The purpose of the present investigation was to determine whether lignin, a component of cabbage fiber, would protect against mammary carcinogenesis by N-methyl-N-nitrosourea (MNU) in Sprague-Dawley rats. A six-week study was conducted using diets containing 0.5-5% dietary wood lignin (a readily available, purified source). These diets were well tolerated by the rats, and a carcinogenesis study using 5 mg MNU/100 g body wt i.v. at 50 days of age was conducted, with the 2.5% lignin diet fed from 6 through 8 weeks of age followed by 5% lignin diet until 20 weeks after MNU. Dietary lignin and MNU treatment increased food consumption (p < 0.05), and body weight was slightly reduced at 10 and 20 weeks after MNU in the MNU-5% lignin diet group (p < 0.05). Serum estradiol was not altered by dietary lignin or MNU treatment, but uterine weights were highest in the MNU-control diet group 4 and 12 weeks after MNU. Expression of creatine kinase B, and estrogen-responsive gene, was lower in eh uteri of the MNU-lignin diet group than in other groups at 20 weeks. Mammary carcinogenesis was not altered by dietary lignin. However, uterine endometrial adenocarcinoma was observed only in the MNU-lignin diet group (4 carcinomas/40 effective rats) (p < 0.05).

We have examined the ability of 17beta-estradiol (E2) to induce development of mammary cancers in the female ACI rat. Continuous treatment with E2, delivered through release from s.c. Silastic tubing implants containing 27.5 mg crystalline hormone, resulted in rapid development of palpable mammary tumors in ovary-intact ACI rats. In a population of 21 E2-treated rats, palpable tumors were first observed following 99 days treatment and 100% of the treated population developed tumors within 197 days. The median and mean times to appearance of first palpable tumor were 143 and 145 days respectively. All mammary tumors were classified as carcinomas and invasive features were observed. Circulating E2 levels in the treated animals at the time of sacrifice averaged 185 pg/ml serum. Mammary tumors were not observed in ovary-intact female ACI rats that were not treated with E2. This is the first report indicating that this naturally occurring estrogen is capable of inducing mammary cancers in the ACI rat strain. Mammary carcinoma did not develop in a population of 11 ovariectomized female ACI rats treated with E2 for a period of 140 days. Circulating E2 levels in the treated ovariectomized animals averaged 207 pg/ml. These data indicate that the ovary modulates estrogen-mediated mammary carcinogenesis in this rat strain. Both ovary-intact and ovariectomized female ACI rats displayed similar susceptibilities to E2-induced pituitary tumors and hyperprolactinemia. Pituitary weight was increased 6.0-fold in ovary-intact ACI rats and 5.3-fold in ovariectomized female rats. Circulating prolactin levels averaged 2318 ng/ml in E2-treated, ovary-intact rats and 2285 ng/ml in E2-treated, ovariectomized ACI rats. These data indicate that estrogen-induced hyperprolactinemia is not the sole factor leading to development of mammary cancers in the E2-treated ACI rat.

Objectives: We evaluated the responses of HIV-infected children to a single dose of split-virus influenza vaccine and the relationship to viral load and other characteristics., Methods: Fifty-three HIV-infected children ages 1.8 to 13.2 years were given influenza vaccine for the 1994 to 1995 influenza season (Wyeth-Ayerst: A/Texas H1N1, A/Shangdong H3N2 and B/Panama). Immunologic and virologic factors were assessed at the time of and 2 to 10 weeks after immunization., Results: The differences between pre- and postimmunization CD4+ counts, CD4+:CD8+ ratios and viral load were not significant. Thirty-one of 53 children (58.4%) had a > 2-fold increase and 16 of 53 (30%) had a 4-fold rise in their postimmunization antibody titers for at least one component of the vaccine. Influenza immunization in the 1993 to 1994 flu season and administration of intravenous immunoglobulin around the time of immunization was not associated with immune response to the vaccine. Factors that were negatively associated with antibody response included increased time between samples (P = 0.004) and decreased preimmunization CD4+:CD8+ ratio (P = 0.02)., Conclusions: Influenza immunization in this population is safe, and a positive antibody response to influenza immunization is not associated with significant clinical events or change in HIV-1 plasma viral burden.

Human immunodeficiency virus type 1 (HIV-1) isolates obtained prior to and during a combination therapy trial comparing zidovudine (AZT; 3'-azidothymidine) monotherapy with AZT plus 2',3'-dideoxyinosine (ddI) or AZT plus 2',3'-dideoxycytidine (ddC) were assessed for the development of drug resistance. Drug susceptibility was measured by using two different phenotypic assays, one that requires infection of peripheral blood mononuclear cells with HIV-1 isolated from cocultures and a second based on infection of HeLa CD4+ cells with recombinant virus containing the reverse transcriptase (RT) of the clinical isolate. In addition, genotypic assessment of resistance was obtained by DNA sequencing of the RT coding region. No difference in the development of AZT resistance was noted in isolates from individuals receiving AZT monotherapy or combination therapy. However, a low frequency of ddI or ddC resistance was seen in isolates from the combination arms, which may at least partially explain the enhanced efficacy observed with these drug combinations compared with monotherapy. It was noted from DNA sequencing that a relatively high frequency of the nonnucleoside RT inhibitor resistance mutation, codon 181 changed from encoding Tyr to encoding Cys, was present in some isolates both before and during nucleoside analog combination therapy. Since these patients were unlikely to have access to nonnucleoside RT inhibitors, it is probable that this mutation preexisted at a reasonable level in the wild-type virus population. Comparisons of the AZT susceptibility assays indicated a good correlation between the phenotypic and genotypic determinations. However, direct numerical comparisons between the phenotypic assays were not reliable, suggesting that valid comparisons of different resistance data sets will require the use of the same assay procedure.

A randomized controlled study was done to determine whether initial combination therapy with zidovudine and zalcitabine or zidovudine and didanosine would delay the emergence of zidovudine-resistant virus. Human immunodeficiency virus (HIV)-1-infected patients with <300 CD4 cells/mm3 and <4 weeks of prior zidovudine therapy were randomized to zidovudine, zidovudine plus zalcitabine, or zidovudine plus didanosine. Combination therapy did not delay the emergence of zidovudine-resistant virus isolates. However, combination therapy resulted in a significant increase in CD4 cells through 72 weeks compared with zidovudine monotherapy and a greater and more sustained decline in serum HIV-1 RNA. Although this trial was not designed as a clinical end-point study, patients assigned to zidovudine plus didanosine combination therapy experienced a significant delay in time to first AIDS-defining event or death compared with those assigned to zidovudine monotherapy.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes numerous immunotoxic effects including thymic involution and an immunosuppression of nonspecific as well as specific cell- and humoral-mediated immunity. TCDD administration to laboratory animals also results in a decreased resistance to numerous bacteria, viruses, and parasites. Effects on virus host resistance appear to be among the most sensitive effects of TCDD immunotoxicity. However, previous studies have not achieved a no effect level. The present studies utilized an influenza virus host resistance model in mice to quantify the sensitivity of this model to TCDD and to determine the NOAEL (no observed adverse effect level) of TCDD for influenza virus. Results indicated that a single dose of TCDD at 0.10, 0.05, or 0.01 microgram/kg resulted in an increased mortality to Hong Kong influenza virus when mice were challenged 7 days after TCDD administration. Increased mortality was not correlated with increased virus titers in the lungs. TCDD at 0.005 or 0.001 micrograms/kg had no effect on influenza-induced mortality. TCDD alone did not affect thymus weight at any dose administered in this study. TCDD also did not alter the virus-enhanced increase in lung weight:body weight ratio nor the virus-induced decrease in thymus weight. Thus, low levels of TCDD exposure lead to enhanced mortality to influenza virus; however, the mechanism of this effect remains to be elucidated. Nonetheless, enhanced mortality to influenza virus in mice following a single dose of 10 ng TCDD/kg represents the most sensitive adverse effect yet reported for TCDD.

We have isolated and sequenced the promoter region of the mouse gene (mOAT) encoding ornithine aminotransferase. A comparison of these mOAT sequences with previously reported sequences for the rat and human genes encoding OAT, rOAT and hOAT, respectively, revealed a 256-bp region flanking the transcription start point that is highly conserved between the three genes. This region contains sequence motifs resembling binding sites for general transcription factors, as well as other trans-acting regulatory proteins.

Purpose: Here we report the results of a phase III study, to evaluate whether the addition of cisplatin to radiation therapy (XRT) would improve progression-free survival or overall survival for patients with locally advanced unresectable non-small-cell lung cancer (NSCLC)., Patients and Methods: Two hundred forty patients with biopsy-proven unresectable NSCLC without distant metastases or lower-stage medically inoperable patients were randomized to one of two treatment arms. Arm A consisted of thoracic XRT alone, 60 to 65 Gy total tumor dose in daily fractions of 1.80 to 2.00 Gy; and arm B consisted of identical XRT with the addition of cisplatin 70 mg/m2 every 3 weeks for three cycles beginning on the first day of irradiation., Results: Two hundred fifteen patients were eligible and assessable. The overall response rate was 50% on the combination arm versus 38% on the XRT-alone arm (P = .076). The median progression-free survival time was 23 versus 22 weeks, respectively (P = .0537). The median survival time was 43 weeks on the combination arm versus 46 weeks on the XRT arm (Poverall = .3469). The 1-, 2-, and 5-year survival rates were 43%, 18%, and 5% on the combination arm versus 45% 13%, and 2% on the XRT arm, respectively., Conclusion: Cisplatin, administered every 3 weeks, does not significantly improve response rate, progression-free survival, or overall survival when added to thoracic XRT for locally advanced unresectable NSCLC.

The purposes of this study were to determine whether expression of the gene encoding ornithine aminotransferase (OAT) in the rat liver and kidney is regulated by retinoic acid (RA) and to characterize further the role of thyroid hormone in regulating the expression of this gene. The level of OAT messenger RNA (mRNA) was reduced 70% in the liver of animals fed a vitamin A-deficient diet relative to that in animals fed a vitamin A-sufficient diet. RA, administered at a dose of 20 micrograms/rat to A-deficient rats for 1 or 3 days, restored OAT mRNA to near the level observed in animals fed the A-sufficient diet. Retinol was also effective in this regard. T3, when injected alone at a dose of 10 micrograms/100 g BW, had no effect on the level of OAT mRNA in the liver. However, when injected concurrently with RA, T3 blocked the ability of RA to induce OAT mRNA in the liver of rats fed the vitamin A-deficient diet. Animals made both vitamin A deficient and hypothyroid responded to RA in a manner similar to vitamin A-deficient animals. The vitamin A-deficient, hypothyroid rats responded somewhat differently to T3, however. T3 was unable to block the induction of OAT mRNA in the liver of vitamin A-deficient, hypothyroid rats when injected concurrently with RA for 1 day, but did block the induction of OAT mRNA by RA when these two hormones were injected concurrently for 3 days. These data indicate that RA and T3 exert opposing effects on the level of OAT mRNA in the liver. The effects of RA and T3 on OAT mRNA were markedly different in the kidney. Neither vitamin A deficiency nor RA had any apparent affect on the level of OAT mRNA in the kidney. T3, however, increased the level of OAT mRNA in the kidney of vitamin A-deficient rats. In the kidney of vitamin A-deficient, hypothyroid rats, T3 was unable to increase OAT mRNA when injected for 1 day, but did increase this mRNA when injected for 3 days. Together, these data indicate cell-type specific effects of both RA and T3 on the OAT gene.

Increasing evidence supports the view that therapy with combinations of antiretroviral drugs provides greater and more sustained benefits in the treatment of HIV infection than either monotherapy or sequential therapy. As the number of licensed and developmental antiretroviral agents grows, in vitro analysis is increasingly being used to aid in the selection of effective combinations. An assay has been designed to ascertain the inhibitory action of drug combinations on HIV-infected MT4 cells, allowing rapid evaluation of those that may be of use in the clinic. Manipulation of this system also provides data on the efficacy of drugs under conditions of high viral load and against resistant strains, providing valuable information for the treatment of antiretroviral-experienced patients with advanced disease.

Ten different three-drug combinations have been analyzed for their ability to prevent HIV-induced cytopathic effects (CPEs) in a continuous human T-lymphoblastoid cell line. Agents acting at the same as well as at different sites in the HIV-1 replicative cycle were used. Each compound was analyzed at peak and trough plasma levels achieved in monotherapy and in the presence of HIV-1 strains 3B and MN at a viral inoculum varying from 1 x TCID50 (50% tissue culture inhibitory dose) to 1,000 x TCID50. Using a viral inoculum of 10 x TCID50 HIV-1 3B, it was determined that triple-drug combinations had greater antiviral activities than the corresponding double-drug combinations, which had greater antiviral activities than zidovudine (AZT) monotherapy. The most consistent triple-drug combination, demonstrating superior activity at all concentrations of virus, was AZT + dideoxyinosine + lamivudine which reduced the AZT IC95 (95% inhibitory concentration) by 208-, 57-, 133-, and 25-fold at of 1,000, 100, 10, and 1 x TCID50 HIV-1 3B, respectively, as compared with the IC95 for AZT monotherapy. For all antiviral regimens tested, higher viral inoculum resulted in less inhibition of viral replication and a higher IC95 for AZT. This observation argues for therapeutic intervention at an earlier stage in HIV infection, when viral burden is lower.

One potential explanation for why renal cell carcinoma is usually poorly responsive to chemotherapy is intrinsic multidrug resistance. Dipyridamole (DP) is one of several agents known to bind to P-glycoprotein and potentially reverse multidrug resistance in vitro, and dosages needed to obtain appropriate levels in vivo appear to be well tolerated. The current study was undertaken to evaluate the combination of vinblastine (VLB) and DP in patients with advanced renal cell carcinoma and no prior chemotherapy. From August 1989 through December 1989, 15 patients with inoperable recurrent or metastatic renal cell carcinoma were treated with VLB 0.2 mg/kg (i.v. slow push) and concurrently received DP75 mg p.o. q.i.d. starting 48 hours before and continuing 48 hours after VLB administration. Courses were repeated every 3 weeks for a maximum of 8 weeks, or until disease progression or undue toxicities ensued. The predominant toxicities seen were mild neurotoxicity and leukopenia. Only 1 patient had grade IV leukopenia, and no lethal toxicities occurred. No objective responses were seen; 2 patients had stable disease for 29 and 30+ months. The median survival was 9 months (range: 2.5-30+). We conclude that at the dose and schedule used in this study, the combination of VLB and DP may be administered with acceptable toxicities, but is ineffective in the treatment of advanced renal cell carcinoma.

Numerous anti-HIV drugs are synthetic analogs of endogenous nucleosides. Therefore it is of interest to see if a facilitated nucleoside transport system exists to mediate their uptake into human immune effector cells that are known HIV targets. Nucleoside permeation and metabolism in lymphocytes, macrophages and bone marrow cells isolated from healthy human volunteers were studied, using uridine as the prototype endogenous nucleoside. There are saturable broad specificity nucleoside transport systems in all three cell types, all of which were inhibited by dipyridamole. The Vmax and Km values for uridine transport were 0.05 +/- 0.01 pmol/sec/10(6) cells and 18.4 +/- 4.2 microM, respectively, for lymphocytes, 0.04 +/- 0.01 pmol/sec/10(6) cells and 25.3 +/- 6.6 microM, respectively, for macrophages, and 0.03 +/- 0.01 pmol/sec/10(6) cells and 90.2 +/- 10.1 microM, respectively, for bone marrow mononuclear cells. Anti-HIV dideoxynucleosides such as azidothymidine (AZT), 2',3'-dideoxycytidine (DDC), 2',3'-dideoxyinosine (DDI), 2',3'-dideoxyadenosine (DDA), and 2',3'-dideoxythymidine (DDT) are not substrates of this nucleotide transport system; hence, little or no drug accumulated inside the cells after 60 sec. Equilibration of cells with uridine or dideoxynucleosides for 2 hr resulted in high levels of cellular uridine and DDA, low levels of cellular AZT, but undetectable levels of the other analogs in all three cell types. Active metabolite levels in lymphocytes as assayed by HPLC correlated with the drug permeation results. Our data demonstrated that DDC, DDI, and DDT are not substrates for the nucleoside transporter and cannot diffuse readily across the cell membrane of human immune effector cells. Future anti-HIV drug development efforts should consider drugs that are substrates of the nucleotide transporter to ensure rapid and complete uptake into target cells.

In the rat, the gene (rOAT) encoding ornithine aminotransferase (OAT) is expressed in all cell types examined; however, regulation of rOAT expression is complex and cell-type specific. Various regions of the rOAT 5' flanking domain were cloned upstream from the cat reporter gene, and the expression of these OAT::cat fusions was examined following transfection into rat kidney epithelial cells (NRK-52E), human embryonic kidney cells (293), and rat hepatoma cells (H-4-II-E). Although these experiments suggested the presence of one or more positive regulatory elements between nucleotides -661 and -158, and one or more negative elements upstream from nt -897, none of these putative elements appeared to function in a cell-type-specific manner. The nt sequence of 2531 bp of the rOAT domain flanking the promoter revealed several putative promoter/enhancer elements in positions analogous to the human OAT gene, numerous AGGTCA-like motifs related to the binding sites for the estrogen and thyroid hormone receptors, and multiple motifs resembling a putative regulatory element associated with genes encoding enzymes of the urea cycle. Finally, sensitivity of the 5' end of rOAT to cleavage by DNase I was examined, as DNase-I-hypersensitive sites (DHS) are often found in association with cis-acting regulatory elements. Two DHS were identified; one DHS approximately 140 bp upstream, and the second DHS approximately 300 bp downstream, of the transcription start point (tsp). These data provide the foundation upon which to base future studies aimed at elucidating the molecular mechanisms through which rOAT expression is regulated in a cell-type specific manner.

We have previously demonstrated that population density alters the responsiveness of GH4C1 pituitary tumor cells to 17 beta-estradiol (E2). At a low population density E2 was observed to increase prolactin mRNA and stimulate cell proliferation, whereas this estrogen was unable to elicit these responses when the cells were maintained at a 4-fold higher population density. In an attempt to determine the mechanism through which population density alters responsiveness to E2, the steady-state level of estrogen receptor (ER), the affinity of ER for E2, and ER down-regulation have been examined in both intact and fractionated cells using ligand binding and ligand exchange assays. Data presented herein demonstrate that (1) GH4C1 cells maintained at low density expressed fewer ER than cells cultured at high density; (2) ER in cells cultured at high density displayed a reduced affinity for E2; (3) ER down-regulation occurring within 1 h of E2 addition appeared to be more pronounced in high density cultures; and (4) steady-state levels of ER were similar in low and high density cells treated with E2 for 1 through 5 days. Although none of these observations appear to correlate with the previously observed effects of population density on the responsiveness of GH4C1 cells to E2, they further illustrate the potential of the culture environment to alter the responsiveness to estrogenic stimuli by altering the properties of the ER.

Herein we describe the isolation and characterization of the rat gene encoding ornithine aminotransferase (rOAT). Six unique genomic clones were characterized and assigned to two nonoverlapping contigs representing approx. 33 kb of the rat genome. The 5' contig contains the rOAT promoter, exons 1 and 3, and a portion of exon 4; an exon corresponding to exon 2 of the human OAT gene (hOAT) was not identified. The rOAT promoter contains several putative regulatory elements in positions similar to hOAT. The 3' contig contains exons 7 through 11 in their entirety. Data presented and discussed herein suggest that approx. 3.0 kb of uncloned genomic DNA, containing the remainder of exon 4 and all of exons 5 and 6, separate the two contigs. Together, these data suggest that rOAT extends over approx. 20 kb and is organized into at least 10 exons, thereby closely resembling hOAT in size and exon/intron organization. Isolation of rOAT provides an important tool for examining the molecular mechanisms through which estrogen and thyroid hormone regulate transcription of this gene in a cell-type specific manner.

From December 1987 through April 1989, 40 patients with extensive-stage small cell carcinoma of the lung were enrolled in a Hoosier Oncology Group (HOG) trial using etoposide, ifosfamide, and cisplatin (VIP). Patients with extensive disease were eligible if they had not received prior chemotherapy, had a Karnofsky performance status of 50 or more, and had adequate renal function (creatinine, less than 1.5 mg/dl) and bone marrow reserve (granulocyte count, greater than or equal to 2500/microliters; platelets, greater than or equal to 125,000/microliters). Doses of therapy were: etoposide 75 mg/m2/day on days 1 to 5, ifosfamide 1.2 g/m2/day on days 1 to 5, and cisplatin 20 mg/m2/day on days 1 to 5. The first 11 patients received a 5-day course; this was repeated every 21 days for four cycles, but therapy was shortened to 4 days when unacceptable toxicity was noticed in these patients. Overall, 14 (37%) had a complete remission (overall response rate, 71.1%) with a median survival of 42 weeks (28 weeks on 5-day regimen and 45 weeks on 4-day regimen). There were five early deaths. Although toxic, VIP produces a high complete remission rate in patients with extensive disease and warrants further evaluation. A prospective randomized trial comparing cisplatin and etoposide to the VIP regimen is underway through HOG.

As a first step towards understanding the molecular mechanisms through which the expression of the gene (OAT) encoding ornithine aminotransferase (OAT) is regulated in a tissue-specific manner, we have used a near full length OAT cDNA to isolate related sequences from a rat genomic DNA library. Twenty-one unique clones representing five contigs and spanning approximately 140 kb of genomic DNA were isolated and characterized. From these clones we have identified a single expressed OAT gene and three processed pseudogenes. The comparison of the EcoRI, BamHI, and HindIII fragments contained within these genomic clones with those detected in total genomic DNA by the cDNA probe suggests that essentially all of the OAT-related sequences in the rat genome have been isolated. Thus, the tissue-specific regulation of OAT gene expression appears to be effected through a single expressed gene. Data are presented which suggest that the OAT-1, OAT-2, and OAT-3 pseudogenes arose approximately 28.5, 7.3, and 25.1 Myr ago, respectively. Mutation rates are presented for each codon position of the expressed rat and human OAT genes. The region of the rat genome flanking the boundary of the OAT-3 pseudogene is of additional interest as it shares considerable identity to sequences contained within expressed genes and flanking other processed pseudogenes.

Twenty-six previously treated patients with refractory small cell lung cancer (SCLC) were entered into a Hoosier Oncology Group phase II trial of daily oral etoposide 50 mg/m2/d. Twenty-five patients had prior exposure to cisplatin plus etoposide, and 14 of the 26 patients (54%) had prior therapy with CAV (cyclophosphamide, doxorubicin, and vincristine). Nonhematologic toxicity was mild; the dose-limiting toxicity was granulocytopenia. One complete response and five partial responses were seen (duration, 6 to 20 weeks), for an overall response rate of 23%. We conclude that daily oral etoposide is a well-tolerated and easily administered drug in refractory SCLC and has definite therapeutic activity.

A patient with refractory acute myeloid leukemia was treated with tiazofurin, an agent that causes inhibition of tumor cell proliferation by depressing GTP concentrations in the malignant cells. The initial dose of 1100 mg/m2 was ineffective clinically and biochemically. Dose escalations to 1650, 2200, and finally 3300 mg/m2 resulted in a marked decrease in the absolute number of blasts without causing bone marrow hypoplasia or marked neutropenia. The decrease in the peripheral blast cell count was observed subsequent to a decline in GTP concentrations in the leukemic cells to less than 30% of the pretreatment value. Consecutive bone marrow examinations showed a remarkable shift from myeloblasts to more mature myeloid elements, suggesting an in vivo differentiative action of tiazofurin. Although a total dose of 23,650 mg/m2 was administered over a 13-day period, only very mild side effects were noted. The absence of complications reported by others in Phase I trials with tiazofurin may be related to our slow administration of the drug by pump over a 1-h period in this trial. Tiazofurin appears to be a promising agent in the treatment of leukemia because of its selective action on leukemic cells and the availability of a rapid in vitro method capable of predicting sensitivity of leukemic cells to the agent and monitoring its activity during treatment by measuring thiazole-4-carboxamide adenine dinucleotide and GTP concentrations. These observations are being tested in a larger group of leukemic patients.

The Hoosier Oncology Group (HOG) treated 13 patients with bronchogenic carcinoma with an innovative schedule of cisplatin and VP-16. Unexpected toxicity was noted, with five deaths secondary to granulocytopenia and septic shock and three episodes of renal failure. Despite early closure of this study, we conclude that this schedule of cisplatin and VP-16 results in greater toxicity than comparable dosages in a more routine schedule.

Cisplatin plus 5-FU appears to have significant additive activity in various tumors, such as head and neck carcinoma and esophageal cancer. A partial explanation for this may be drug synergism, which has been noted in the L1210 leukemia model. Based on these data, a prospective trial of weekly bolus 5-FU (15 mg/kg) and cisplatin (60 mg/m2) given every 3 weeks was initiated at Indiana University. Forty-one patients, of whom 38 are fully evaluable for response, were treated with these two drugs. Ten partial and one complete response (complete + partial response rate = 29%) were observed in the 38 evaluable patients. Thirteen additional patients had stable disease for greater than or equal to 3 months. The median durations of remission and survival time were 6 and 10.3 months, respectively. Myelosuppression was unusually severe, with granulocyte counts less than 1000/mm3 in 65% of patients, including four patients with granulocyte count nadirs less than 100/mm3. Three patients developed granulocytopenic fever, with two drug-related deaths (sepsis, hyperosmolar coma). Nearly all patients had nausea and vomiting, but this was not a treatment-limiting toxic effect in any patient. Although this combination suggests a higher response rate than usually seen with bolus iv 5-FU in colon cancer, a trial comparing 5-FU alone or with cisplatin to determine whether true synergy exists is currently underway.

Photographic processing techniques for producing high efficiency, low noise phase holograms on silver halide emulsions have been investigated. Control of the emulsion and maintenance of the integrity of the holographic record throughout all processing were identified as prime requisites for high quality holographic reconstructions. Holograms have been obtained which yield approximately 30% diffraction efficiency and signal to scattered noise (S/N) in excess of 50:1 in the reconstruction of a diffuse wave-front. Holographic interferometry and ghost imaging with diffuse subjects has also been achieved with these techniques.

Defect enhancement in semiconductor wafers is shown to be possible by spatial frequency filtering without the necessity for filter alignment. The technique depends on the wafer being sufficiently periodic. Coherent optical and digital computer simulation results are presented.

Multicolor image reconstruction from holograms behaving as two-dimensional media suffer severely from overlap of the similarly structured crosstalk images unless special techniques are used in formation. This paper describes examples of two techniques which eliminate the crosstalk images. The first technique involves nonoverlapping spatial multiplexing and is illustrated by interlacing on the hologram the colored images of a mask placed in the reference beam. The second technique involves coding the reference beam so that each colored reference wave varies in a unique manner over the hologram plate. Photographs of the resulting multicolor reconstructions are shown.