Tumor heterogeneity and in vitro chemosensitivity testing in ovarian cancer

OBJECTIVE: Our purpose was to study the heterogeneity of drug response in fresh human ovarian tumors to chemotherapeutic agents in an in vitro chemosensitivity assay. STUDY DESIGN: This assay evaluates total tumor cell kill by measuring the intracellular adenosine triphosphate levels of untreated controls and drug-exposed cells at various doses after culture for 6 days. The surviving fraction is calculated by dividing the treated values with the control values. One hundred tumors were tested against four single drugs (cisplatin, the active metabolite of cytoxan, 4-hydroxyperoxy-cyclophosphamide, Taxol, and carboplatin) and two drug combinations (cisplatin plus 4-hydroxyperoxy-cyclophosphamide; cisplatin plus Taxol). RESULTS: There is great variation in the degree of cell death for single drugs and drug combinations among the 100 tumors tested. CONCLUSION: More effective clinical response to chemotherapy may be achieved in patients with ovarian cancer by selecting the most active drugs for chemotherapy, on the basis of in vitro chemosensitivity test results for individual patients.     

Clinical criteria of chemosensitivity test

Chemosensitivity test is useful in evaluating the appropriate cancer chemotherapy for solid tumors which are less sensitive to conventionally available antitumor agents. Most of assays used in clinics are in vitro assay, in which the cutoff concentration and contact time should be determined to mimic the clinical response of the chemosensitivity test. Since the antitumor effect of most of antitumor agents depends on their concentration x contact time, and the contact time will be determined according to the assay, the cutoff concentration will be calculated from the cumulative efficacy curve or 50% inhibitory concentration and in vivo response of human tumor xenografts in nude mouse. The clinical result obtained from chemosensitivity test was discussed.     

Differential chemosensitivity in oncogene-transformed cells

The effects of anticancer drugs on cell growth have been investigated by MTT colorimetric assay using mouse and rat cultured cells transformed by various oncogenes and tumor viruses. Aclarubicin, mitomycin C and 1-hexylcarbamoyl-5-fluorouracil showed higher growth-inhibitory activities toward transformed cells than those toward the normal counterparts. Nimustine, bleomycin and 5-fluorouracil also showed selective growth-suppressive activities toward transformed cells except for a few cell lines. ras-oncogene-transformed cells were more sensitive toward 5-fluorouracil and 1-hexylcarbamoyl-5-fluorouracil than the normal parent cells and other transformed cells. These drugs would thus be effective in the chemotherapy of ras-induced cancer.     

RB protein status and chemosensitivity in non-small cell lung cancers

A total of 1,207 patients received a Medtronic Jewel active can ICD (models 7218C, 7219C), with a Transvene lead in 97 centers in Europe and North America. Nineteen implants were from the right pectoral region. Patients with right-sided ICDs did not differ in terms of mean age, % male, left ventricular ejection fraction, New York Heart Association Functional Class, antiarrhythmic drug therapy, indication for the implantable cardioverter defibrillator, and R wave values at implantation, but tended to have slightly higher pacing thresholds (1.2 +/- 0.5 V vs 1.0 +/- 0.6 V, P = 0.012) and higher defibrillation thresholds (14.7 +/- 6.4 J vs 11.5 +/- 6 J, P = 0.11) compared with patients with left sided implants. Patients with right-sided implants had a longer implantation time compared with patients with left-sided implants (118 +/- 70 minutes vs 91 +/- 46 minutes, P = 0.074). In follow-up, 5 patients with right-sided implantation received successful therapy for either ventricular fibrillation, (8 episodes) or ventricular tachycardia (5 episodes). No ineffective therapy from the device was delivered in any patients with right-sided implantation. Right-sided pectoral implants are feasible with the Medtronic Jewel active can ICD.     

Quality control and factor VIII assays

We report the results of the treatment of 23 patients with macrodactyly. Eighteen had a two-stage bulk-reducing (defatting) procedure; phalangectomy was used to shorten the digits. At a mean follow-up of nine years (2 to 12), two patients had been lost to follow-up, and three await a second-stage procedure. Good cosmetic correction was achieved in 12 patients, with satisfactory results in seven; two patients had poor results and required amputation.     

In vitro drug assays and statistical analysis

The development of in vitro drug tests to assess the efficacy of drugs against Pneumocystis carinii has been hindered by the lack of efficient methods for continuous cultivation of the microorganism. However, different short-term culture systems have been proposed by many teams. In the present contribution an in vitro microplate drug assay and two statistical programs allowing the analysis of results are presented.     

Convenient colorimetric and fluorometric assays for S-nitrosothiols

S-nitrosothiols have been shown to affect a number of physiological functions. Several techniques have been used to detect these species in biological systems, primarily by methods utilizing chemiluminescence. Since the apparatus required for measurement of chemiluminescence are not readily available in most laboratories, methods employing more conventional techniques such as uv-vis and fluorescence spectroscopy may be of greater use. Herein, we report the development of colorimetric and fluorometric methods for the reliable quantitation of S-nitrosothiols. Solutions containing sulfanilamide/N-(1-naphthyl)- ethylenediamine dihydrochloride or 2,2'-azinobis (3-ethylbenzthiazoline-6-sulfonic acid), when exposed to S-nitrosoglutathione (GSNO), S-nitrosocysteine, or S-nitrosoacteylpenicillamine, resulted in no absorbance changes in the range of 400-800 nm. Exposure to HgCl2 or Cu(acetate)2 resulted in release of nitric oxide (NO) from the S-nitrosothiols. The liberated NO reacted subsequently with oxygen and formed a chemical species which reacted with either analysis solution, resulting in an increase in absorption between 400 and 800 nm. A plot of RSNO versus absorbance was linear for both mercury(II) and copper(II) ions where the slope in the presence of mercury ion was significantly greater than that for copper ion. The sensitivity was as low as 5 microM RSNO using HgCl2. The fluorometric method using 2, 3-diaminonaphthalene as the scavenger of the NOsolidusO2 products gave a sensitivity of 50 nM for GSNO. In addition, S-nitrosylated proteins were quantitated using the fluorometric technique. These methods provide accurate determination of low concentrations of S-nitrosothiols, utilizing conventional spectroscopic techniques available in most laboratories.     

Methods for investigation and estimation of individual chemosensitivity of tumors (review)

A review of the current methods for the estimation of tumor sensitivity to antiblastomic drugs is presented. Methods for the in vitro and in vivo cultivation of tumor cells are discussed. Among them the following methods are reviewed: the method for the cultivation of tumor heterotransplants in diffusion chambers and its various modifications, the method for the cultivation of human tumors under the kidney capsule, the method for the cell cultivation in primary short-term suspensions, the methods using enriched media, the methods for the cell cultivation on acetate cellulose fibres, in monolayers, etc.     

Channel electrophoresis for kinetic assays

A rectangular channel electrophoresis system and a cylindrical sampling capillary combination allows chemical changes in nanoliter-volume samples to be monitored as a function of time. The electrophoretic microseparation is carried out in a rectangular channel with a 7 -cm-long, 40-microm x 2.5-cm geometry and is coupled to a 50-microm-i.d. cylindrical sample introduction capillary. The channel width dimension is used as a time axis by moving the outlet of the sampling capillary across the entrance of the separation channel. Detection of the separated analyte bands is achieved with laser-induced fluorescence and spatially resolved detection based on a charge-coupled device. The system is characterized with a series of fluorescein thiocarbamyl amino acid derivatives; limits of detection are < 10(-8) M for amino acids and 10(-9)M (425 zmol) for fluorescein. The ability to achieve a time-based dynamic microseparation is demonstrated by monitoring fluorescent product formation during the enzyme-catalyzed hydrolysis of fluorescein di-beta-D-galactopyranoside (FDG), a commonly used fluorescent substrate for enzymological studies.     

Relation between chemosensitivity and the ventilatory response to exercise in chronic heart failure

OBJECTIVES: This study sought to establish the chemosensitivity of patients with chronic heart failure. BACKGROUND: The ventilatory response to exercise is often increased in patients with chronic heart failure, as characterized by the steeper regression slope relating minute ventilation to carbon dioxide output. We hypothesized that the sensitivity of chemoreceptors may be reset and may in part mediate the exercise hyperpnea seen in this condition. METHODS: Hypoxic and peripheral hypercapnic chemosensitivity were studied in 38 patients with chronic heart failure (35 men, 3 women; mean [+/-SE] age 60.2 +/- 1.3 years; radionuclide left ventricular ejection fraction 25.7 +/- 2.3%) and 15 healthy control subjects (11 men, 4 women; mean age 54.9 +/- 3.0 years) using transient inhalations of pure nitrogen and single breaths of 13% carbon dioxide, respectively. The change in chemosensitivity during mild exercise (25 W) was assessed in the first 15 patients and all control subjects. Central hypercapnic chemosensitivity was also characterized in 25 patients and 10 control subjects by the rebreathing of 7% carbon dioxide in 93% oxygen. Cardiopulmonary exercise testing was performed in all subjects. RESULTS: Maximal oxygen consumption was 16.6 +/- 0.9 versus 29.7 +/- 2.2 mol/kg per min (p < 0.0001), and the ventilation-carbon dioxide output regression slope was 37.2 +/- 1.5 versus 26.5 +/- 1.4 (p < 0.0001) in patients and control subjects, respectively. Hypoxic and central hypercapnic chemosensitivity were enhanced in patients (0.707 +/- 0.076 vs. 0.293 +/- 0.056 liters/min per % arterial oxygen saturation [SaO2], p = 0.0001 and 3.15 +/- 0.41 vs. 2.02 +/- 0.25 liters/min per mm Hg, p = 0.025, respectively) and correlated significantly with the ventilatory response to exercise. Hypoxic chemosensitivity was augmented during exercise in patients and in control subjects but remained higher in the former (1.530 +/- 0.27 vs. 0.685 +/- 0.12 liters/min per %SaO2, p = 0.01). The peripheral hypercapnic chemosensitivity of patients at rest and during exercise was similar to that in control subjects, consistent with its lesser contribution to overall carbon dioxide chemosensitivity. CONCLUSIONS: Enhanced hypoxic and central hypercapnic chemosensitivity may play a role in mediating the increased ventilatory response to exercise in chronic heart failure.     

Diagnostic assays in cytomegalovirus retinitis

The aim of the work is to inspect the influence of the treatment by using hyper-correcting prisms on the vertical deviations of the eyes and on the head's position in persons with nystagmus. We observed 4 persons with nystagmus without strabismus and 3 persons with convergent squint. In persons without strabismus the prismatic correction placed with an edge in the direction of the "calm's zone" (quiet's zone) to obtain the straight position of the head when looking forwards was applied. Twice a day during 10 minutes the patients were making the movement's exercises in the vertical and horizontal direction looking by the prism separately by each eye. This prism (often 35 D prism) was placed with the edge in the direction of greater deviation of the oblique inferior muscles and the left rectus inferior muscle. Patients with convergent strabismus were treated according to the principles of localization method with consideration of the localize exercises by using hyper-correcting prisms in the vertical and horizontal directions. Two patients had a surgery in order to eliminate not aesthetic and strong prisms which were applied because of large horizontal squint. One patient with convergent alternate squint with hyperactivity of both inferior oblique muscles and inferior rectus muscle of the left eye was treated without surgery, only by the conservative treatment with prisms. In all patients we obtained a straight position of the head despite of the nystagmus still existing during the eyes movements in some directions. The treatment by using hyper-correcting prisms can completely replace the surgical treatment or is able to supplement it and prevent relapses.     

Quality control of multidrug resistance assays in adult acute leukemia: correlation between assays for P-glycoprotein expression and activity

We have compared multiple assays for the P-glycoprotein (Pgp/MDR1) phenotype in fresh and thawed adult acute leukemia to validate and quantitate measures for the expression and function of Pgp. The results are related to the Pgp-expressing KB8 and KB8-5 call lines. The most sensitive assay was the measurement of modulation of the rhodamine 123 (R123) fluorescence by 2 micromol/L PSC833, followed by the modulation of the probe calcein-AM. We also found a good intralaboratory and interlaboratory correlation between the values of the R123/PSC833 assay for fresh as well as thawed samples. In addition, the affects of PSC833 on 3H-daunorubicin (DNR) accumulation, DNR fluorescence, and 3H-vincristine accumulation were very similar. The correlation between the DNR/PSC833 and R123/PSC833 test was r = .86 (N = 51). The modulation of drug accumulation by 8 micromol/L verapamil was the some as the PSC833 effect for DNR (117%, N = 21), but was higher for vincristine in every single case (161% v 121%, N = 22; P< .001), indicating additional verapamil effects, not related to Pgp. The correlation of the staining of viable cells for Pgp with the monoclonal antibody MRK16 was r = .77 (N = 52) for the R123/PSC833 functional test and r = .84 (N = 50) for the DNR/PSC833 test. From these results it could be calculated that a maximal increase of the mean DNR accumulation of about 50% can be achieved by blocking Pgp pump activity with PSC833 in leukemic blast samples with the highest mean Pgp expression. Subpopulations of blast calls with higher Pgp activity are likely to be present. Their relevance has to be studied further. The methods outlined here allow the reliable, quantitative monitoring of the Pgp/MDR1 phenotype in leukemias in multicentered, clinical Pgp modulation studies.