Harry K. Slocum

Effect of Amphotericin B and Micafungin Combination on Survival, Histopathology, and Fungal Burden in Experimental Aspergillosis in the p47phox−/− Mouse Model of Chronic Granulomatous Disease

ABSTRACT Chronic granulomatous disease (CGD) is an inherited disorder of the NADPH oxidase characterized by recurrent life-threatening bacterial and fungal infections. We characterized the effects of single and combination antifungal therapy on survival, histopathology, and laboratory markers of fungal burden in experimental aspergillosis in the p47phox−/− knockout mouse model of CGD. CGD mice were highly susceptible to intratracheal Aspergillus fumigatus challenge, whereas wild-type mice were resistant. CGD mice were challenged intratracheally with a lethal inoculum (1.25 × 104 CFU/mouse) of A. fumigatus and received one of the following regimens daily from day 0 to 4 after challenge (n = 19 to 20 per treatment group): (i) vehicle, (ii) amphotericin B (intraperitoneal; 1 mg/kg of body weight), (iii) micafungin (intravenous; 10 mg/kg), or (iv) amphotericin B plus micafungin. The rank order of therapeutic efficacy based on prolonged survival, from highest to lowest, was as follows: amphotericin B plus micafungin, amphotericin B alone, micafungin alone, and the vehicle. Lung histology showed pyogranulomatous lesions and invasive hyphae, but without hyphal angioinvasion or coagulative necrosis. Treatment with micafungin alone or combined with amphotericin B produced swelling of invasive hyphae that was not present in mice treated with the vehicle or amphotericin B alone. Assessment of lung fungal burden by quantitative PCR showed no significant difference between treatment groups. Serum galactomannan levels were at background despite documentation of invasive aspergillosis by histology. Our findings showed the superior efficacy of the amphotericin B and micafungin combination compared to either agent alone after A. fumigatus challenge and also demonstrated unique features of CGD mice as a model for experimental aspergillosis.

HER-2/neu Protein Expression and Gene Alteration in Stage I-IIIA Non-Small-Cell Lung Cancer: A Study of 140 Cases Using a Combination of High Throughput Tissue Microarray, Immunohistochemistry, and Fluorescent In Situ Hybridization

Regarding HER-2/ neu expression (gene or protein level) in lung cancer, several studies with inconsistent results have been recently reported, partially due to variable techniques used and/or heterogeneous populations examined. The objective of this study was to examine HER-2/ neu expression in a well-defined cohort of non–small-cell lung cancers (NSCLC) and in nonneoplastic lung tissue utilizing a combination of high-density tissue microarray, immunohistochemistry (IHC), and fluorescent in situ hybridization (FISH) under uniform test conditions. One hundred forty stage I-IIIA primary NSCLCs and 38 non-neoplastic lung samples were examined. IHC, using an FDA-approved Hercept monoclonal antibody kit, was performed and HER-2/ neu gene alteration was assessed by FISH. The association of expression of HER-2/ neu with clinicopathologic parameters was analyzed. Ninety-four percent of tumor samples (131/140) were fully interpretable after tissue processing. Twenty-five of them (19%) overexpressed (2+, 3+) HER-2/ neu, while 106 (81%) had no or weak expression. All thirty-four interpretable non-neoplastic lung samples were negative for HER-2/ neu alteration at protein and gene level. HER-2/ neu protein overexpression correlated well with HER-2/ neu gene amplification (r =.83, P < 0.001). HER-2/ neu overexpression was significantly associated with histologic subtype: 19 adenocarcinomas (19/82, 23%) versus 4 squamous cell carcinomas (4/44, 9%) overexpressed Her-2/ neu (P = 0.04). Statistical significance was observed between HER-2/ neu expression and tumor differentiation, with strong positive (3+) expression observed more frequently in poorly differentiated tumors (P = 0.01). Patients with HER-2/ neu abnormalities, particularly HER-2/ neu gene amplification, exhibited a shorter survival (P = 0.043). The statistically significant difference (P < 0.005) between HER-2/ neu alteration in tumor samples(25/131, 19%) and in the nonneoplastic tissue (0/34, 0%) implies that HER-2/ neu may have a role in the carcinogenesis of NSCLC. The findings provide evidence supporting the hypothesis that the HER-2/ neu receptor may represent a useful molecular target in the treatment of NSCLC. The significant association of HER-2/ neu expression and gene amplification with poorly differentiated carcinoma compared with well differentiated carcinoma suggests that HER-2/ neu may be involved in NSCLC tumor evolution. Patients with HER-2/ neu gene amplification and strong positive expression of HER-2/ neu protein showed a strong tendency toward shorter survival.

Combined action of paclitaxel and cisplatin against wildtype and resistant human ovarian carcinoma cells

Purpose: The combination of paclitaxel (PTX) and cisplatin (DDP) shows good clinical efficacy against ovarian cancer. In order to examine the potential cellular basis for this, and provide leads as to how to optimize the combination, we examined the role of sequence of exposure to PTX and DDP on cell growth in vitro. Methods: Four human ovarian carcinoma cell lines, A121, A2780/WT, A2780/DX5B and A2780/CP3, two human head and neck carcinoma cell lines, A253 and FaDu, and the human ileocecal carcinoma cell line, HCT-8, were treated with PTX + DDP with seven schedules: (A) 96 h exposure to PTX + DDP; (B) 24 h PTX alone, then 72 h PTX + DDP; (C) 4 h DDP alone, then 92 h PTX + DDP; (D) 24 h PTX alone, 4 h DDP alone, then 68 h drug-free; (E) 4 h DDP alone, 24 h PTX alone, then 68 h drug-free; (F) 3 h PTX alone, 1 h DDP alone, then 92 h drug-free; and (G) 1 h DDP alone, 3 h PTX alone, then 92 h drug-free. Each of 66 two-drug experiments included five plates (440 randomly treated wells per experiment). Cell growth was measured by the sulforhodamine B assay. The nature and the intensity of the drug interactions were assessed by fitting a seven-parameter model to data with weighted nonlinear regression, enabling the estimation of an interaction parameter, α, with its standard error. Results: Overall there was very little departure from Loewe additivity: 43 experiments showed Loewe additivity, 10 showed Loewe antagonism, and 13 showed slight Loewe synergy. In vitro Loewe synergy was rare, was small when present, and reproducible only for the A121 and HCT-8 cells exposed to schedule D (24 h PTX prior to 4 h DDP). Isobolographic analysis showed complex combined-action surfaces with regions of local Loewe synergy and antagonism. Conclusion: It appears unlikely that the good clinical efficacy of the combination is primarily caused by a synergistic interaction at the cellular level.

PARABOLIC GROWTH PATTERNS IN 96-WELL PLATE CELL GROWTH EXPERIMENTS

SummaryIn preparing for the routine use of the ubiquitous in vitro cell growth inhibition assay for the study of anticancer agents, we characterized the statistical properties of the assay and found some surprising results. Parabolic well-to-well cell growth patterns were discovered, which could profoundly affect the results of routine growth inhibition studies of anticancer and other agents. Four human ovarian cell lines, A2780/WT, A2780/DX5, A2780/DX5B, and A121, and one human ileocecal adenocarcinoma cell line, HCT-8, were seeded into plastic 96-well plates with a 12-channel pipette, without drugs, and grown from 1–5 d. The wells were washed with a plate washer, cells stained with sulforhodamine B (SRB), and dye absorbance measured with a plate reader. Variance models were fit to the data from replicates to determine the nature of the heteroscedastic error structure. Exponential growth models were fit to data to estimate doubling times for each cell line. Polynomial models were fit to data from 10-plate stacks of 96-well plates to explore nonuniformity of cell growth in wells in different regions of the stacks. Each separate step in the assay was examined for precision, patterns, and underlying causes of variation. Differential evaporation of water from wells is likely a major, but not exclusive, contributor to the systematic well-to-well cell growth patterns. Because the fundamental underlying causes of the parabolic growth patterns were not conclusively found, a randomization step for the growth assay was developed.

Assessment of Human Genitourinary Tumors and Chemosensitivity Testing In 3-Dimensional Collagen Gel Culture

A recently described collagen gel culture technique has been modified to evaluate the growth characteristics and chemosensitivity patterns of genitourinary neoplasms. Fresh human surgical explants incorporated radiolabeled DNA precursors [H3)thymidine or deoxyuridine) in 97% of 38 patient specimens (18/19 bladder, 3/3 prostate, 2/2 testis, 13/14 renal), after being maintained for three to 13 weeks/passage, with several specimens reaching their sixth passage (20 months). Control cellular DNA incorporation ranged from five to 90% (#cells labeled/#cells evaluated), with median labeling for bladder 30%, prostate 80%, testis 90%, and renal 80%. Original histopathologic classification was maintained in all cases. Tumor volume and glucose consumption were other measurable parameters. Seventy-three surgical specimen cultures were treated with chemotherapeutic agents after a minimum of four weeks in culture. Single agent exposures were 24 hours at 1X and 10X reported peak plasma concentrations. Combination agents were sequenced as in current clinical protocol for bladder tumors and fourteen day continuous fluorodeoxyuridine (FdURD) exposure for renal tumors. Sensitivity was found in 1/2 prostate and 1/10 renal tumors to Adriamycin, 8/15 bladder and 1/2 testis tumors to cisplatin, 11/28 renal tumors to FdURD and 6/16 bladder tumors to MVAC combination chemotherapy. This culture system offers the advantages of in vivo-like solid tumor growth, a high culture success rate, longevity in culture, maintenance of the primary histopathology and reproducible chemosensitivity response.

Involvement of cyclin D1-cdk5 overexpression and MCM3 cleavage in bax-associated spontaneous apoptosis and differentiation in an A253 human head and neck carcinoma xenograft model.

Time‐dependent ladder‐type DNA fragmentation and morphological alterations consistent with apoptosis were observed among A253 human head and neck squamous cell carcinoma (HNSCC) cells in nude mice from 15 to 18 days after transplantation, without any drug treatment. No evidence of ladder‐type DNA fragmentation was detected in A253 cells in vitro or in normal nude mouse tissues (skin and muscle). Our aim was to explore molecular factors associated with such spontaneous apoptosis. Bcl‐2 protein expression decreased, while bax protein expression increased from day 9 after transplantation. Moreover, altered expression of bcl‐2 and bax was accompanied by the increased proteolytic cleavage of poly(ADP‐ribose) polymerase (PARP). Time‐dependent dephosphorylation of Rb, followed by proteolytic cleavage, was also observed from day 9 after transplantation. The data indicate that the caspase‐3 activation and cleavage of Rb protein may represent important steps in the regulation pathway of bax‐mediated spontaneous apoptosis. Interestingly, the time‐dependent activation of spontaneous apoptosis was almost simultaneous with the induction of differentiation and increased expression of several differentiation‐associated regulatory proteins. An increased expression of cyclin D1 and cyclin‐dependent kinase‐5 (cdk5) was observed from day 9 after transplantation, whereas only slight alteration of cdk4 expression was found. The time‐dependent activation of cyclin D1 and cdk5 preceded both the induction of ladder‐type DNA fragmentation and increased keratin pearl formation. Furthermore, MCM3 was cleaved early in spontaneous apoptosis and differentiation. Our observations suggest the involvement of cyclin D1‐cdk5 overexpression and MCM3 cleavage in bax‐mediated spontaneous apoptosis and differentiation in A253 xenografts. P53 and WAF1 proteins were not expressed in the xenografts, indicating that the changes in the regulatory proteins during apoptosis and differentiation were not p53 or WAF1 dependent. Int. J. Cancer 83:341–348, 1999.

Cellular heterogeneity in DNA damage and growth inhibition induced by ICI D1694, thymidylate synthase inhibitor, using single cell assays

Heterogeneity in the response of the HCT-8 (human ileocecal adenocarcinoma) tumor cell line to a new thymidylate synthase inhibitor, ICI D1694, was investigated in terms of induction of DNA single-strand breaks and cytotoxicity, applying the single cell alkaline gel (SCG) electrophoresis assay and the individual colony formation assay (iCFA), respectively. ICI D1694 induced maximal total DNA single-strand breaks 24 hr after a 2-hr drug exposure with incomplete repair by 72 hr. The level of DNA damage was concentration dependent and paralleled cellular growth inhibition in vitro. The proportion of cells with DNA damage and the extent of DNA single-strand breaks increased with drug concentration. At 1 microM ICI D1694 (IC95), a significant level of DNA damage was detected in 58% of the cells; however, 25% of the cells had little or no damage. Using the iCFA system, it was observed that with 1 microM ICI D1694, only 2.6% of the seeded cells maintained a colony growth rate similar to that of the control colonies, and 22% of the cells were growing significantly more slowly. In conclusion, the SCG assay and the iCFA identified subpopulations of cells that were unaffected by ICI D1694. Although these cells represented only a small proportion of the total cell population, this phenomenon of heterogeneity in response to ICI D1694 might limit its therapeutic efficacy.

Folic acid-enhanced synergy for the combination of trimetrexate plus the glycinamide ribonucleotide formyltransferase inhibitor 4-[2-(2-Amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4]thiazin-6-yl)-(S)-ethyl]-2,5-thienoylamino-l-glutamic acid (AG2034): Comparison across sensitive and resistant human tumor cell lines

Folic acid (PteGlu)-enhanced intense synergy has been observed between nonpolyglutamylatable dihydrofolate reductase (DHFR) inhibitors and polyglutamylatable inhibitors of other folate-requiring enzymes, such as glycinamide ribonucleotide formyltransferase (GARFT) and thymidylate synthase. Since this phenomenon is potentially therapeutically useful, we explored its universality by examining the combined action of a DHFR inhibitor, trimetrexate (TMQ), with a GARFT inhibitor, 4-[2-(2-amino-4-oxo-4,6,7,8-tetrahydro-3H-pyrimidino[5,4,6][1,4]++ +thiazin-6-yl)-(S)-ethyl]-2,5-thienoylamino-L-glutamic acid (AG2034), in eight human cultured cell lines. Using a 96-well plate cell growth inhibition assay, four ileocecal adenocarcinoma cell lines [HCT-8, HCT-8/DW2 (Tomudex-resistant), HCT-8/DF2 (Tomudex-/FdUrd-resistant), and HCT-8/50 (adapted to 50 nM PteGlu)], three head and neck carcinoma cell lines [A253, FaDu, and Hep-2/500 (FdUrd-resistant)], and a non-small cell lung carcinoma cell line [H460] were treated for 96 hr with TMQ + AG2034 in the presence of 23 or 40 microM PteGlu. Cell growth was measured with the sulforhodamine B assay at the end of this period. Drug interactions were assessed by fitting a 7-parameter model including a synergism parameter, alpha, to data with weighted nonlinear regression. Isobologram analysis was also applied. At 23 microM PteGlu, cells exhibited similar intensities of Loewe synergy for the combination of TMQ + AG2034. Loewe synergy was abolished in HCT-8/50 cells cultured and studied in 50 nM PteGlu. At 40 microM PteGlu, the intensity of the combined action in all cell lines was increased However, the most intense Loewe synergy was seen with HCT-8, HCT-8/DF2, H460, FaDu, A253, and Hep-2/500 cells, whereas the HCT-8/50 subculture showed less of the phenomenon, and PteGlu enhancement was the least with HCT-8/DW2, a subline deficient in folylpolyglutamate synthetase (FPGS). The universality of the PteGlu-enhanced intense synergy phenomenon is suggested. Impaired FPGS activity and low-folate adaptation prior to treatment significantly lessen the degree of PteGlu enhancement.

Implications for Clinical Pharmacodynamic Studies of the Statistical Characterization of an In Vitro Antiproliferation Assay

AbstractModeling of nonlinear pharmacodynamic (PD) relationships necessitates the utilization of a weighting function in order to compensate for the heteroscedasticity. The structure of the variance was studied for concentration–effect data generated in an in vitro 96-well plate cell growth inhibition assay, where data are numerous (480 data points per experiment) and replication is easy. From the five candidate models that were considered, the power function