Enzyme-induced posterior vitreous detachment in the rat produces increased lens nuclear pO2 levels.

It has been proposed that disruption of normal vitreous humor may permit O(2) to travel more easily from the retina to the center of the lens where it may cause nuclear cataract (Barbazetto, I.A., Liang, J., Chang, S., Zheng, L., Spector, A., Dillon, J.P., 2004. Oxygen tension in the rabbit lens and vitreous before and after vitrectomy. Exp. Eye Res. 78, 917-924; Harocopos, G.J., Shui, Y.B., McKinnon, M., Holekamp, N.M., Gordon, M.O., Beebe, D.C., 2004. Importance of vitreous liquefaction in age-related cataract. Invest. Ophthalmol. Vis. Sci. 45, 77-85). In the present study, we injected enzymes intravitreally into guinea pigs (which possess an avascular retina) and rats (which possess a vascular retina) to produce either vitreous humor liquefaction plus a posterior vitreous detachment (PVD) (with use of microplasmin) or vitreous humor liquefaction only (with use of hyaluronidase), and 1-2 weeks later measured lens nuclear pO(2) levels in vivo using a platinum-based fluorophore O(2) sensor (Oxford-Optronix, Ltd.). Experiments were also conducted in which the animals were allowed to breathe 100% O(2) following intravitreal injection with either microplasmin or hyaluronidase in order to investigate possible effects on O(2) exchange within the eye. Injection of guinea pigs with either of the two enzymes produced no significant differences in lens pO(2) levels 1-2 weeks later, compared to controls. However, for the rat, injection of microplasmin produced a 68% increase in O(2) level in the center of the lens, compared to the controls (5.6mm Hg increasing to 9.4mm Hg, p<0.05), with no corresponding effect observed following similar use of hyaluronidase. Treatment of guinea pigs with microplasmin dramatically accelerated movement of O(2) across the vitreal space when the animals were later allowed to breathe 100% O(2) (for example, O(2) traveled to a location directly behind the lens 5x faster than control; p<0.01); however, the effect following treatment with hyaluronidase was significantly less. When microplasmin-injected rats breathed 100% O(2), the time required for O(2) to reach the center of the lens was 3x faster than control (0.4 min compared to 1.4 min, p<0.01). The results have implication with regard to the occurrence of age-related PVD in the human, and a possible acceleration of maturity-onset nuclear cataract. In addition, enzymatic creation of a PVD to increase the rate of O(2) exchange within the vitreal space may have potential application for treatment of retinal ischemic disease.

Subclonal heterogeneity of the multidrug resistance phenotype in a cell line expressing antisense MDR1 RNA.

A multidrug resistant (MDR) cell line was transfected with an antisense MDR1 expression vector and transfectant clones were analyzed for reversion of the MDR phenotype. Only one of 10 antisense-expressing transfectants showed a reduction in drug resistance, MDR1 mRNA and P-glycoprotein. Observations made using rhodamine-123, a fluorescent substrate for P-glycoprotein, revealed that dye retention in individual cells was highly variable within this antisense-expressing clone. Subpopulations were established from the original clone based on differences in rhodamine-123 retention. Rhodamine-123 retention varied inversely with levels of P-glycoprotein and MDR1 mRNA. All subpopulations expressed similar levels of antisense MDR1 RNA yet had dramatic differences in MDR1 mRNA levels. Analysis of vector integration site restriction fragment length polymorphisms confirmed that all populations originated from the same transfectant clone. Nuclear run-on analysis indicated that the mdr1 gene is transcribed at the same rate in all populations, suggesting that the reduction in MDR1 mRNA is mediated posttranscriptionally. Cells with the greatest reduction in MDR1 mRNA accumulate distinct antisense RNA transcripts in the nuclear RNA fraction, suggesting that antisense effectiveness in this system is associated with a nuclear event or process. These results reveal that antisense RNA activity is not necessarily distributed equally within a clonal population.

New immunohistochemical "sandwich" staining method for mdr1 P-glycoprotein detection with JSB-1 monoclonal antibody in formalin-fixed, paraffin-embedded human tissues.

We have developed a new immunoperoxidase "sandwich" staining method for amplified detection of P-glycoprotein (Pgp) that is suitable for use on formalin-fixed, paraffin-embedded (conventional) tissue sections. This was accomplished by substantially changing the procedure described by Chan (1988) so as to increase specific staining intensity and to decrease nonspecific background staining. To determine the most appropriate primary antibody for the assay, we compared the immunoreactivity of JSB-1, C494, and C219 monoclonal antibodies recognizing internal epitopes of Pgp, and MRK16 and 4E3 monoclonal antibodies recognizing external epitopes of Pgp. Paraffin sections of Pgp-positive normal human tissues (adrenal, liver, kidney, and brain), of renal tumors, and of cell pellets of sensitive and multidrug resistant human tumor cell lines (MCF-7, KB) were used for comparisons. Immunostaining was excellent with JSB-1, moderate with C494, and very weak with C219. MRK16 and 4E3 showed no reaction. Nonspecific background staining was reduced by 1) omitting immunoglobulin G from secondary antibodies; 2) decreasing the concentration of peroxidase-antiperoxidase complex; and 3) utilizing casein solution for blocking and washing. Pretreatment of sections before immunostaining was also simplified. Using JSB-1, the threshold for detection of elevated Pgp corresponded to less than two-fold relative resistance to doxorubicin. Applying this method, we found two of 26 non-small cell lung cancers were positive for Pgp, consistent with previous results of others using frozen sections. This new immunoperoxidase sandwich staining method using JSB-1 now allows reliable Pgp detection in sections of formalin-fixed, paraffin-embedded (archived) surgical specimens and small biopsy materials commonly used for diagnostic purposes.

Hypoxia-induced sympathetic inhibition of the fetal plasma insulin response to hyperglycemia.

Large-for-delivery date babies, considered characteristic of diabetic pregnancy, are believed to result from fetal hyperinsulinemia. Paradoxically, infant birth weights tend to be low-for-delivery date in mothers with more severe diabetes. We tested the hypothesis that hypoxemia in such fetuses leads to sympathoadrenal stimulation and inhibition of insulin secretion; and, thus, produces a net reduction in the growth-promoting effects. Fetal sheep were prepared with chronic peripheral and adrenal cannulas. Fetal blood gases, lactate, norepinephrine, and epinephrine secretion rates; and plasma norepinephrine, glucose, and immunoreactive insulin concentrations were determined at 30-min intervals during a 2-h baseline period and a 4-h period of hyperglycemia divided into 2-h segments of hypoxemia (with and without alpha-blockade) and hyperoxia. Hypoxemia-hyperoxia sequences were varied randomly. Well-oxygenated fetuses responded to a threefold increase in glucose with a sixfold increase in plasma immunoreactive insulin. With hypoxemia, norepinephrine and epinephrine secretion were elevated and the insulin response was blocked. With hypoxemia and phentolamine blockade, the insulin response was enhanced with a 10-fold increase above baseline. In severe maternal diabetes with vascular disease or with poor control and very high glucose levels, the fetus is likely to be relatively hypoxemic. Our experiments suggest that in this situation, the fetal insulin response to hyperglycemia will be attenuated; this effect is mediated, at least partly, through sympathoadrenal stimulation.

Reversal of C1300 murine neuroblastoma multidrug resistance by cremophorEL, a solvent for cyclosporin A.

We previously developed a homoharringtonine resistant C-1300 neuroblastoma cell line with cross-resistance to adriamycin and increased levels of p-glycoprotein, and showed that drug resistance could be reversed in this cell line by cyclosporin A. The present study shows that cremophor EL, a parenteral vehicle for cyclosporin A, can also completely reverse this multidrug resistance in a clonogenic assay system. Cremophor EL incubated with resistant cells for up to six days did not reduce levels of p-glycoprotein. Intracellular homoharringtonine analysis using HPLC revealed increased drug accumulation in resistant cells treated with cremophor EL. The increased drug level was not due to blocking of drug efflux commonly seen in other multidrug resistant models. The data suggest that resistance modulation with cyclosporin A should be interpreted with caution when cremophor EL is a solvent. Our work suggests cremophor EL, a relatively nontoxic lipophylic solvent, may have a direct effect on membrane permeability, although other mechanisms cannot be ruled out.

Adrenal corticosteroid secretion in fetal sheep: pulsatile pattern at rest.

This study was undertaken to define the resting pattern of fetal pituitary-adrenocortical function. Experiments were performed at 127-145 days gestation in fetal sheep with chronic peripheral and adrenal cannulas inserted under halothane anesthesia. With the fetus in a baseline state, over 6 h, at 30-min intervals, maternal and fetal peripheral samples were collected for blood gases and cortisol (F), corticosterone (B), and adrenocorticotropic hormone (ACTH) concentrations, and three successive, 2-min adrenal samples were collected for determination of F and B secretion rates. We observed high-frequency, episodic bursts of F secretion. A lower frequency oscillation of F secretion, with a period of approximately 90 min, was defined by cosinor analysis. The mean amplitude of the oscillation increased from 45 to 507 ng/min with advancing gestation. The pattern of B secretion was similar to that for F but was quantitatively lower. An oscillatory period of approximately 90 min for plasma F was present in a majority of experiments. Pulsatile rhythms for ACTH were defined in 10 of 14 experiments, with periods ranging from 1.64 h in the least mature group to 2.37 h in the oldest fetus. Mean data revealed exponential increases in both F secretion and plasma ACTH from 129 to 145 days gestation.

Comparison of an immunoperoxidase "sandwich" staining method and western blot detection of P-glycoprotein in human cell lines and sarcomas.

The applicability of a multilayer immunoperoxidase "sandwich" method (IpS) developed by Chan14 for the amplified detection of P-glycoprotein (Pgp) was investigated. The authors examined 15 formalin-fixed cell lines, as well as formalin-fixed, paraffin-embedded sections from single biopsies of 46 sarcomas. The cell lines included sensitive and multidrug resistant sublines (KB, A2780, MCF-7, HeLa) with various relative degrees of resistance to doxorubicin (Dox). The sarcoma biopsy specimens were selected on the basis of the results obtained in Western blot (WB) detection of Pgp (22 positive and 24 negative by WB) using C219 and C494 monoclonal antibodies to Pgp. The IpS method employed C219. The least resistant cell line in which Pgp could be detected by IpS was fivefold resistant to doxorubicin, whereas Pgp was detected by WB in cells greater than twofold resistant. Cell lines having greater than fivefold resistance to Dox were positive by both IpS and WB analyses. The less resistant cell lines contained more nonreactive cells whereas the highly resistant cell lines showed more homogeneous strong membrane reactions. Among the six cell lines determined to be Pgp negative by WB analysis, no false positive immunostaining by IpS existed. One of 22 WB positive and 7 of 24 WB-negative sarcoma biopsy specimens were positive by IpS methods. Reaction varied and was always focal (a minimum of 3-5 cells, ranging up to 3-4 high power fields) indicating pronounced heterogeneous distribution of Pgp. Thus, WB can detect low average (overall) levels of Pgp in tumor samples but such low concentrations of PgP at the single cell are not detectable by IpS methods. However, IpS can discern among many Pgp-negative cells small subpopulations of immunoreactive cells, which are not detected by WB analysis due to Pgp dilution by the membrane protein of numerous Pgp negative cells. IpS and WB used together as complementary methods can provide more complete information about Pgp distribution and content, particularly in the case of heterogeneous human tumors. The IpS method is more suitable for less drastically treated (not embedded) cell line specimens than for paraffin-embedded (routine) sections. Some modification of the present IpS protocol seems necessary to increase its sensitivity and reduce the disparity with WB results.

Enhanced sensitivity to G418 of human KB cells adapted to certain media and sera.

Sensitivity to the neomycin derivative G418 was determined for a human cell line, KB 3-1, that had been adapted to six different combinations of media and sera. The results indicate that while the plating efficiency is similar for all conditions, the susceptibility to G418 can differ markedly depending on the particular combination of media and sera used. This suggests that in experiments using neomycin resistance as a selectable marker, conditions may be found where the amount of G418 required for selection and maintenance of transfected cell lines can be reduced, providing a significant savings.

Resistance of CHO cells expressing P-glycoprotein to cyclopropylpyrroloindole (CPI) alkylating agents.

Several new antitumor agents belonging to the class of minor groove binders that are able to form covalent bonds with DNA via a cyclopropylpyrroloindole (CPI) group are susceptible to a multidrug resistance (MDR) phenotype in Chinese hamster ovary (CHO) cells. The multidrug resistant CCHR-C5 cell line was 16-, 23- and 13-fold more resistant to the analogs U-73,975, U-77,779 and U-80,244, respectively, although its cytotoxic response to the parent compound CC-1065 was similar to the response of the drug-sensitive wild-type cells (AuxB1). For a sequence of MDR cell lines showing increasing expression of P-glycoprotein (Pgp) there were corresponding increments in the level of resistance to U-73,975, arguing that Pgp is the key determinant in resistance of the MDR cells to CPI agents. MDR cells treated with U-73,975 showed diminished generation of covalent adducts on DNA as well as increased resistance to cytotoxicity.

Evaluation of a self-help dietary intervention in a primary care setting.

BACKGROUND: Dietary intervention to reduce fat consumption and increase fiber consumption has been recommended by the National Cancer Institute, but there is little evidence concerning the effectiveness of self-help materials. The purpose of this study was to evaluate such self-help materials, introduced by a nurse in a primary care setting. METHODS: A randomized controlled trial involving 242 subjects was conducted in two primary care clinics in Chapel Hill, NC, in 1987. Changes in fat and fiber consumption in the intervention and control groups during the 3-month interval between interviews were compared using analysis of covariance. RESULTS: The estimated reduction in fat was 3.8g larger for the intervention group than for the control group, but the confidence interval included zero. For those individuals who had some responsibility for meal preparation there was a larger difference (-6.9g) in favor of the intervention group, although the difference using calorie-adjusted values was -3.8g with a 95% confidence interval (-7.1, -0.4). The differences for fiber change were smaller. CONCLUSIONS: We found significant small but consistent differential changes associated with a minimal self-help intervention, but we cannot rule out the possibility of some response bias. Nonetheless, this study demonstrates that the use of self-help materials for dietary change is feasible, and may be effective.

Modulation of drug resistance in homoharringtonine-resistant C-1300 neuroblastoma cells with cyclosporine A and dipyridamole.

The development of resistance accounts for therapy failure in the majority of advanced cases of neuroblastoma in children. A new transplantable murine C-1300 neuroblastoma cell line was developed in vitro, by repeated exposure of a sensitive cell line to increasing, but sublethal, doses of Homoharringtonine (HHT). The ED50 of the highly resistant cells for HHT, using a standard agar colony assay, is 480 ng/ml, compared with 13 ng/ml for the sensitive parental line. The resistant cells have cross-resistance to a number of other agents, including adriamycin, vinca alkaloids, melphalan, and CCNU. Western blot analysis revealed progressive increases in P-glycoprotein, parallel to the graded development of resistance with a 29-fold elevation in the highest resistant cells. High-performance liquid chromatography (HPLC) indicated that resistant cells have a significantly lower uptake of HHT than parental sensitive cells. cyclosporine A (CsA) and dipyridamole (DPM) could modulate the acquired resistance and completely restore the cytotoxic effects of HHT and adriamycin as determined by the clonogenic assay. The reversal of resistance by CsA and DPM was dose dependent. With the relative low toxicity of dipyridamole and CsA in doses required for modulation of resistance, these agents may be candidates for clinical utilization in chemotherapy of resistant neuroblastoma.

Induction of heat-labile sites in DNA of mammalian cells by the antitumor alkylating drug CC-1065.

CC-1065 is a very potent antitumor antibiotic capable of covalent and noncovalent binding to the minor groove of naked DNA. Upon thermal treatment, covalent adducts formed between CC-1065 and DNA generate strand breaks [Reynolds, R. L., Molineux, I. J., Kaplan, D.J., Swenson, D.H., & Hurley, L.H. (1985) Biochemistry 24, 6228-6237]. We have shown that this molecular damage can be detected following CC-1065 treatment of mammalian whole cells. Using alkaline sucrose gradient analysis, we observe thermally induced breakage of [14C]thymidine-prelabeled DNA from drug-treated African green monkey kidney BSC-1 cells. Very little damage to cellular DNA by CC-1065 can be detected without first heating the drug-treated samples. CC-1065 can also generate heat-labile sites within DNA during cell lysis and heating, subsequent to the exposure of cells to drug, suggesting that a pool of free and noncovalently bound drug is available for posttreatment adduct formation. This effect was controlled for by mixing [3H]thymidine-labeled untreated cells with the [14C]thymidine-labeled drug-treated samples. The lowest drug dose at which heat-labile sites were detected was 3 nM CC-1065 (3 single-stranded breaks/10(6) base pairs). This concentration reduced survival of BSC-1 cells to 0.1% in cytotoxicity assays. The generation of CC-1065-induced lesions in cellular DNA is time dependent (the frequency of lesions caused by a 60 nM treatment reaching a plateau at 2 h) and is not readily reversible. The induction of heat-labile sites in cellular DNA was confirmed by gel electrophoretic analyses of the damage to intracellular simian virus 40 (SV40) DNA in SV40-infected BSC-1 cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of auromomycin-resistant hamster cell mutants that display a multidrug resistance phenotype.

We have selected and characterized Chinese hamster ovary (CHO) cells resistant to auromomycin (AUR), an antitumor antibiotic composed of a protein moiety and a nonpeptide chromophore. AUR is cytotoxic as a consequence of DNA strand-scission activity associated with the chromophore. Initial single-step selections for clones resistant to AUR detected a subpopulation of phenotypically resistant colonies, but nearly all such clones failed to display heritable resistance. One isolate that did show somewhat increased resistance was selected further and yielded a clone designated AURR-R1 that exhibits stable 10-fold increased resistance to AUR. The R1 line is also resistant to the AUR chromophore and cross-resistant to the closely related agent neocarzinostatin (NCS) and to the NCS chromophore. For AUR-treated whole cells, resistance to AUR cytotoxicity was inversely correlated with DNA damage as measured by filter elution; by contrast, isolated nuclei from sensitive and resistant cells displayed similar levels of AUR-induced DNA damage. The R1 cell line was found to be cross-resistant to colchicine, Adriamycin, Daunomycin, and vinblastine. The resistance phenotype is expressed with incomplete dominance in cell hybrids and appears similar to the "classic" multidrug resistance of CHO cells selected with other agents. Indeed, we found the multidrug-resistant CHO line CCHR-C5 to be about 5-fold cross-resistant to AUR and to NCS. We ascertained that AUR-resistant (AURR) isolates express elevated levels of the molecular weight 170,000 P-glycoprotein often associated with multidrug resistance and also contain amplified DNA sequences that contain the gene for P-glycoprotein. When multiple-step enrichment selections were carried out as an alternative approach for isolating AURR mutants, each of nine clonal isolates showed phenotypes resembling the AURR-R1 line. Thus, our findings imply that increased cellular resistance to AUR may frequently be associated with P-glycoprotein-mediated multidrug resistance.

Elevated pentose cycle and glucuronyltransferase in daunorubicin-resistant P388 cells.

Anthracycline resistance of P388 daunorubicin-resistant cells cannot be accounted for merely by differences in drug uptake and retention; protection against intracellular drug was also indicated. Cytotoxicity of daunorubicin may be partially due to the formation of free radicals and reactive oxygen species (hydrogen peroxide, hydroxyl radical, singlet oxygen, and superoxide anion radical). Protection against free radicals and peroxides is largely dependent upon the availability of reduced glutathione, which in turn requires NADPH for its continual regeneration. Pentose phosphate cycle (also called hexose monophosphate shunt) is known to provide NADPH for maintenance of glutathione. Activities of the two NADPH-producing dehydrogenases of the cycle, glucose-6-phosphate and 6-phosphogluconate dehydrogenase, were 40% higher (P less than 0.05) and activity of the cycle in intact cells was 2-fold higher in the resistant than the sensitive cells. The cycle was as active in these cells as it is known to be in macrophages, indicating a very effective protection against oxidative stress, free radicals, and alkylating electrophiles. Elevated activity of the pentose phosphate pathway in drug-resistant cells can represent a mechanism of resistance against multiple structurally unrelated drugs. Efflux of daunorubicin may be aided by further metabolism to glucuronides. Daunorubicinol, a known active metabolite of daunorubicin, can be metabolized to a glucuronide by the cells and eliminated into the surrounding medium. Glucuronidation of daunorubicinol was evidenced by (a) release of daunorubicinol following glucuronidase hydrolysis of media from cell incubations with 1.8 microM daunorubicin and (b) production of radioactive glucuronide when cell homogenates were incubated with UDP-[14C]glucuronic acid plus daunorubicinol. Glucuronyltransferase activity with a broad substrate specificity was found in the cells. Using model substrates, 1-naphthol and o-aminophenol, it was determined that glucuronyltransferase activity was 4 times higher in daunorubicin-resistant than -sensitive P388 cells. Elevated glucuronyltransferase could contribute to daunorubicin and multidrug resistance.

The relationship between multidrug resistance and tumor necrosis factor resistance in an EL4 cell line model.

A number of recent studies have implied that a relationship exists between cellular sensitivities to tumor necrosis factor (TNF) and expression of the classic multidrug resistance (MDR) phenotype. However, different conclusions have been reported concerning whether TNF sensitivity is positively or negatively correlated with MDR (Hong, W.-S.; Sijo, N.; Sasaki, Y.; Shinkai, T.; Eguchi, K.; Sakurai, M.; Takamashi, H.; Nakano, H.; Nakagawa, K.; Twentyman, P. R. Jpn. J. Can. Res. (Gann) 78:1274-1280; 1987 and Dollbaum, C.; Creasey, A. A.; Dairkee, S. H.; Hiller, A. J.; Rudolph, A. R.; Lin, L.; Vitt, C.; Smith, H. S. Proc. Natl. Acad. Sci. USA 85:4740-4755; 1988). An apparent relationship of TNF sensitivity to P-glycoprotein (P-170gp) mediated MDR was investigated in EL4 murine T-lymphoma cell lines sensitive and resistant to Adriamycin (ADM). No consistent association was found between MDR and TNF responses when the lines were subcloned. Whereas the MDR phenotype of subclones (as assessed by ADM resistance and P-170gp expression) reflected that of the cell line from which they were derived, the TNF sensitivity of subclones varied widely. Also consistent with independence of P-170gp mediated MDR and TNF response, the P388/ADM cell line (exhibiting P-170gp mediated MDR) remained as resistant to TNF as the P388 parental line. In addition, no evidence was found of modified recognition of MDR EL4 cell lines by host defense effector cells, and gamma-interferon failed to enhance the susceptibility of either parental or MDR cell line to TNF. These results may be of value in considering therapeutic studies using the ADM/TNF combination treatment.

Improving the adequacy of Pap smears.

Several of the risk factors and pathophysiologic mechanisms for cervical carcinoma implicate the transformation zone of the cervix as the most important area for cytologic sampling. Improved screening can be achieved by ensuring that the transformation zone has been sampled, as evidenced by the number of endocervical cells on the smear. Use of the endocervical brush, combined with feedback from the laboratory about inadequate smears, may improve the technique for obtaining Pap smears.

P-glycoprotein in human sarcoma: evidence for multidrug resistance.

Overexpression of an immunologically conserved, cell-surface glycoprotein (P-glycoprotein) is consistently associated with multidrug resistance in cell lines in vitro. A preliminary survey of specimens from 12 solid tumor types in our laboratories indicates significant overexpression of P-glycoprotein in some sarcomas. When tested by immunoblotting with monoclonal antibodies directed against P-glycoprotein; tumors from six of 25 sarcoma patients displayed elevated levels of P-glycoprotein. Three of the sarcoma patients exhibiting P-glycoprotein had not previously been exposed to chemotherapy, implying that overexpression of this marker and possible concomitant multidrug resistance may not depend only on selection during prior drug treatments. The P-glycoprotein overexpression in the sarcoma specimens is evidence for the presence of multidrug resistant cells in these tumors; thus, our data suggest that this mode of resistance may have clinical significance in sarcoma patients.