Specificity of the control of tumor formation by the blastocyst.

An assay to determine the mechanism of regulation of embryonal carcinoma cells by the blastocyst, which is based on a comparison of tumors produced when the cancer cells are cloned alone or after incorporation into blastocysts, was refined by labeling embryonal carcinoma cells with fluorescent microspheres and by following their fate after injection into the blastocysts. Through the use of the new techniques, it was observed that cells of one line of nullipotent embryonal carcinoma were controlled at the 50% level, those from another were not controlled, and those from a multipotent but undifferentiated line were controlled in almost absolute fashion. Single Sarcoma 180 of L1210 leukemia cells were not controlled when injected into the blastocele, but C1300 neuroblastoma cells were partially controlled. None of these tumors have a normal cellular counterpart in the blastocyst, as does embryonal carcinoma, but neurulation follows blastulation by only a few days, so that the neuroblastoma cells may be regulated at that time. Parietal yolk sac carcinoma cells, which have a counterpart in the late blastocyst, were not controlled. On the basis of these data, it is postulated that, if one embryonic field can regulate its closely related cancer, then there may be an embryonic field capable of regulating each carcinoma.

Constitutive activation of stimulatory guanine nucleotide binding protein (G(S)alphaQL)-mediated signaling increases invasiveness and tumorigenicity of PC-3M prostate cancer cells.

An abnormal stimulation of cAMP signaling cascade has been implicated in various human carcinomas. Since the agents activating G(S)alpha-mediated signaling pathways have been shown to increase in vitro proliferation of prostate cancer cells, present studies examined the G(S)alpha-mediated signaling in tumorigenicity and invasiveness of PC-3M prostate cancer cells. PC-3M cells were stably transfected with plasmids containing either wild type (G(S)alpha-WT) or constitutively active (gsp mutant of G(S)alpha or G(S)alpha-QL) cDNAs. The stable transfectants were then tested for: (1) colony formation in soft agar; (2) cell migration and penetration of basement matrix in an in vitro invasion assay; and (3) the ability to form tumors and metastases in nude mice. PC-3M cells expressing G(S)alpha-QL protein displayed 15-fold increase in their ability to migrate and penetrate the basement membrane as compared to parental PC-3M cells or those expressing G(S)alpha-WT. G(S)alpha-QL transfectants also displayed a dramatically greater rate of growth in soft agar, and greater tumorigenicity and metastasis forming ability when orthotopically implanted in nude mice. All mice receiving PC-3M cells produced primary tumors within 5 weeks after implantation. However, the cells expressing G(S)alpha-QL displayed a significantly faster tumor growth as assessed by prostate weight (greater than 20-fold as compared to PC-3M cells), and produced metastases in kidneys, lymph nodes, blood vessels, bowel mesentery and intestine. Interestingly, expression of G(S)alpha-WT reduced the ability of PC-3M cells to form tumors in nude mice. These results suggest that persistent activation of G(S)alpha-mediated signaling cascade can dramatically accelerate tumorigenesis and metastasizing ability of prostate cancer cells.

Highly loaded and thermally stable Cu-containing mesoporous silica-active catalyst for the NO + CO reaction.

We report the synthesis of a highly loaded and thermally stable Cu-containing mesoporous silica, which was developed by making use of poly(acrylic acid) (Pac) assembled with surfactant (C(16)TAB), as template. On this backbone, TEOS and Cu(II) hydrolysis takes place leading to the development of the final mesostructure. Poly(acrylic acid) is used not only as a micelle structural component but also as a complexation agent for Cu(II) species resulting in high metal loading and increased thermal stability of the mesoporous network. The original uncalcined material possesses hexagonal ordering, while upon calcination it is transformed into a wormlike mesoporous network with metal loading >14 wt % Cu. An evaluation of its performance as heterogeneous catalyst in NO reduction by CO shows catalytic activity comparable with that of noble metal catalysts. Complete NO conversion, with >90% selectivity to N(2), was achieved between 190 and 200 degrees C. The material retained its structure and catalytic activity after 24-h testing at the maximum catalytic conversion of NO and CO.

Inhibition of estrogen stimulated mitogenesis by 3-phenylacetylamino-2,6-piperidinedione and its para-hydroxy analog.

3-Phenylactetylamino-2,6-piperidinedione (A10) inhibited estradiol stimulated cell growth in the MCF-7 (E3) human breast tumor cell line in vivo and in vitro. While high concentrations of A10 were needed to inhibit cell proliferation (IC50 = 3 x 10(-3) M in vitro), the compound demonstrated little toxicity. The effect appeared specific since a hydrolysis product of A10, phenylacetylglutamine, demonstrated no growth inhibitory activity at similar concentrations in MCF-7 (E3) cells in vitro. A computer designed analog, p-hydroxy A10, was more potent than A10 in inhibiting activity in MCF-7 (E3) cells in vitro. The IC50 for p-hydroxy A10 was 7 x 10(-6) M which was comparable to that of the antiestrogen, tamoxifen (IC50 1 x 10(-7) M). All three compounds caused a decline in estrogen receptor levels in a dose-dependent fashion. A10 also inhibited estradiol induction of progesterone receptors. Examination of protein kinase activity following an acute exposure to a 10(-11) M growth stimulatory dose of estradiol revealed a 168% increase in protein kinase activity over that of untreated control cells. A10 in a dose-responsive fashion inhibited the estradiol stimulated increase in protein kinase activity. The protein kinase activity was also inhibited by p-hydroxy A10. These activities of A10 and p-hydroxy A10 coupled with the low toxicity and novelty of the basic A10 structure provide an exciting possibility of developing a new class of clinically useful antineoplastic drugs with minimal side effects.

Flow cytometric analysis of induced human graafian follicles. I. Demonstration and sorting of two luteinized cell populations.

STUDY OBJECTIVE: To test the hypothesis that induced human graafian follicles consist of different steroidogenic cell types on the basis of their light scatter characteristics as determined by flow cytometry. DESIGN: Cross-sectional, observational study. SETTING: Flow cytometry laboratory. PATIENTS: Thirty-six follicular aspirates from nine consecutive patients undergoing in vitro fertilization for tubal factor infertility were evaluated. RESULTS: Two distinct luteal cell populations were recovered. Both populations were positive by Oil Red O staining, suggesting the presence of intracellular lipid. Neither population stained positively for the presence of HLe-1/CD45, an antigen present on all human leukocytes. CONCLUSIONS: Cellular heterogeneity exists within the granulosa cell compartment.

Identification of a low molecular weight polypeptide of pregnant bovine uterine origin (LMW-UDF): influence on coagulation system in vitro. Part I.

Acidified extracts of pregnant bovine uterus were found to contain a heparin-affinity polypeptide(s). The eluted peak was assayed in the Chandler loop method for whole blood and fibrin thrombus generation. Significant differences in the mean weight and size of whole blood thrombi (243.5 +/- 50 mg and 3.5 cm +/- 0.6 cm) occurred with 1 microgram of purified polypeptide when compared to control samples containing albumin (47.87 +/- .30 g and 0.8 cm +/- 0.3 cm). This activity was detected by the Chandler loop method with 100 ng to 10 micrograms of protein when analyzed in a dose-response fashion. When tested for fibrin thrombus formation this activity persisted. Experiments with 125I fibrinogen revealed no net increase or decrease of uptake into whole blood thrombi when purified polypeptide was added. The heparin-affinity polypeptide(s) possessed a molecular weight of approximately 6-4 kDa when examined by SDS-PAGE. We have therefore designated this polypeptide as low molecular weight-uterine derived factor (LMW-UDF).

Ganglioglioma, a malignant tumor? Correlation with flow deoxyribonucleic acid cytometric analysis.

This study describes the flow cytometric deoxyribonucleic acid (DNA) analysis of a resected ganglioglioma. The initial histopathological analysis revealed a benign tumor characterized by a predominance of mature ganglion cells. The flow cytometric DNA analysis of the necrotic areas, however, demonstrated an aneuploid population of cells. Further examination by histological analysis of the tumor revealed both benign and atypical foci. The retrospective DNA analysis performed from paraffin sections of tissue with benign-histological findings demonstrated euploid populations of cells consistent with a benign, slow-growing lesion. In contrast, DNA analysis performed from tissue with atypical histological findings revealed aneuploid populations of cells consistent with a malignant phenotype. Our analysis provides additional data supporting the existence of tumor progression in some gangliogliomas. Results support the concept of tumor cell heterogeneity and the importance of adequate tumor sampling. The finding of aneuploid populations with unfavorable histology further supports the use of flow cytometry as an adjunct method in assessing tumor biology.

Use of verapamil to enhance the antiproliferative activity of BCNU in human glioma cells: an in vitro and in vivo study.

The authors have evaluated the antiproliferative activity of verapamil, alone or in combination with 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) in brain-tumor cells. These effects were studied in vitro using four human glioma cell lines and in vivo using glioblastoma multiforme cells transplanted to athymic nude mice. The results showed that verapamil when used alone produced inhibition of tumor growth; however, when verapamil was used in combination with BCNU (in vitro), significant dose-dependent suppression of proliferation occurred in all four cell lines. The in vivo results were far more dramatic. Mice treated with BCNU (25 mg/kg) plus verapamil (50 mg/kg) achieved a 200-fold decrease in tumor growth with a greater than 80% regression in tumor size. Complete cures were achieved in 80% of the mice observed for at least 50 days following the completion of therapy. These findings support the use of verapamil in overcoming drug resistance in malignant brain tumors.

Far-field responses to stimulation of the cochlear nucleus by microsurgically placed penetrating and surface electrodes in the cat.

OBJECT: A new generation of penetrating electrodes for auditory brainstem implants is on the verge of being introduced into clinical practice. This study was designed to compare electrically evoked auditory brainstem responses (EABRs) to stimulation of the cochlear nucleus (CN) by microsurgically implanted surface electrodes and insertion electrodes (INSELs) with stimulation areas of identical size. METHODS: Via a lateral suboccipital approach, arrays of surface and penetrating microelectrodes with geometric stimulation areas measuring 4,417 microm2 (diameter 75 microm) were placed over and inserted into the CN in 10 adult cats. After recording the auditory brainstem response (ABR) at the mastoid process, the CN, and the level of the inferior colliculus, EABRs to stimulation of the CN were recorded using biphasic, charge-balanced stimuli with phase durations of 80 microsec, 160 microsec, and 240 microsec at a repetition rate of 22.3 Hz. Waveform, threshold, maximum amplitude, and the dynamic range of the responses were compared for surface and penetrating electrodes. The EABR waveforms that appeared for both types of stimulation resembled each other closely. The mean impedance was slightly lower (30 +/- 3.4 kohm compared with 31.7 +/- 4.5 kohm, at 10 kHz), but the mean EABR threshold was significantly higher (51.8 microA compared with 40.5 microA, t = 3.5, p = 0.002) for surface electrode arrays as opposed to penetrating electrode arrays. Due to lower saturation levels of the INSEL array, dynamic ranges were almost identical between the two types of stimulation. Sectioning of the eighth cranial nerve did not abolish EABRs. CONCLUSIONS: Microsurgical insertion of electrodes into the CN complex may be guided and monitored using techniques similar to those applied for implantation of surface electrodes. Lower thresholds and almost equivalent dynamic ranges indicate that a more direct access to secondary auditory neurons is achieved using penetrating electrodes.

Chemotherapy of human carcinoma xenografts during pulsed magnetic field exposure.

Immune deficient mice growing xenografts of HT-29 or A-431 cell lines were treated with cisplatin, carboplatin or doxorubicin in combination with one hour of wholebody pulsed magnetic field (PMF) exposure (calculated peak field 5.2 mTesla, with an average field strength of 0.525 mTeslarms; pulses rose for 120 microseconds and then abruptly fell to neutral, and were repeated at a rate of 250 pulses per second). At 24 days, the mice in each experiment were found to have significantly (p < 0.05, ANOVA) different tumor sizes among groups. The smallest mean tumor volume was consistently found in the drug+PMF group. With A-431 tumors, the cisplatin+PMF group (T) was significantly smaller, 52% [1-(100T/C)], than the cisplatin alone group (C). In HT-29 tumors, those treated with carboplatin+PMF had the smallest tumor volume at just 34% of the carboplatin-alone group. In HT-29 tumors, the doxorubicin+PMF group was 35% of the doxorubicin alone group.

Flow cytometry shows recurrent aneuploid clone after remission in megakaryocytic leukemia.

Flow cytometry using a DNA label can quantitate aneuploid clones in malignant tissue. We illustrated the clinical value of this technique in a 71-year-old woman with acute megakaryocytic leukemia, which was diagnosed by staining of the blasts with factor VIII antigen and their morphologic resemblance to megakaryoblasts. Marrow cells were removed from needle biopsies by vortexing in RPMI medium, centrifuged in Ficoll-Hypaque, stained with a propidium-iodide/NP-40 mixture, and analyzed at 488 nm using an argon laser. During 3 weeks of low-dose cytosine arabinoside (ara-c) infusion therapy, hyperdiploid peak A dropped from 35% (day 0) to 2.3% (day 14) to 0% (day 21), with development of marrow hypoplasia. Similarly, hyperdiploid peak B, went from 7.6% to 9.1% to 3.5%. Subsequently, her marrow recovered normal morphology and lost the aneuploid peaks. Her blood counts recovered to near normal. Four months later, she relapsed and had a return of the day-21, incompletely eradicated peak B. There was no evidence of peak A. Repeated treatment with ara-c resulted in temporary suppression of the disease, but she died 3 months later with progressive hepatosplenomegaly. Analysis of cells from her enlarged liver, heavily infiltrated with blasts, showed a large hyperdiploid peak B. In this patient, ara-c therapy induced a remission with permanent eradication of one clone, but incomplete eradication of a second clone, which ultimately led to her relapse and death.

The proliferative fraction in lymph nodes: a comparison of proliferating cell nuclear antigen morphometry to flow cytometry.

A monoclonal antibody to proliferating cell nuclear antigen (PCNA/cyclin) has recently become available. This antibody, as opposed to Ki-67, can be used on formalin-fixed, paraffin embedded tissue sections and allows retrospective comparison of PCNA positivity to percent S + G2 + M of the cell cycle. To compare fresh lymphoma deoxyribonucleic acid (DNA) analysis with PCNA activity on fixed, paraffin-embedded sections, prospective flow cytometric studies of cell cycle analysis were performed on lymph nodes removed from 10 patients for diagnosis. Six patients had T-cell lymphoma, two had B-cell lymphoma, and two were benign. Using the peroxidase-antiperoxidase method, the nuclear positivity of archival lymphoma cases was also examined. To quantify PCNA positivity, a unique morphometric method was employed that utilized digital imaging by high definition television and ELAS (Earth Land Application Software), a geoscience software used extensively for color quantitation of remote sensed data. The immunologic percent PCNA positivity was 26.1 +/- 20 vs. percent S + G2 + M by flow cytometry of 22.4 +/- 10 with a correlation coefficient (r) of 0.55. This r-value compared favorably to data generated for Ki-67 in solid malignant neoplasms. The six more concordant cases had a percent PCNA positivity of 26.5 +/- 10.0 and a percent S + G2 + M of 27.3 +/- 8.6, r = 0.96. Our study is unique in that it compared fresh lymphoma DNA analysis data with paraffin PCNA data. It is our conclusion that immunologic PCNA positivity in paraffin sections correlates with fresh flow cytometric S + G2 + M in lymph nodes, although careful attention must be paid to the area of the node quantified for PCNA.

Enhanced expression of c-myc and H-ras oncogenes in Letterer-Siwe disease. A sequential study using colorimetric in situ hybridization.

Tissues from two patients with disseminated histiocytosis X (Letterer-Siwe disease) in which histiocytosis X cells exhibited histologic and cytologic features of malignancy were evaluated by in situ hybridization with the use of biotinylated nucleic acid probes to c-myc and H-ras oncogenes. Enhanced expression of these oncogenes was observed in mononucleated and multinucleated cells of histiocytosis X in the terminal proliferative phase but not in the early quiescent phase of Letterer-Siwe disease in both patients. Our findings indicate that deregulation of c-myc and H-ras in histiocytosis X are late events that likely confer a selective growth advantage to histiocytosis X cells.

Embryonal rhabdomyosarcoma in ascitic fluid. Immunocytochemical and DNA flow cytometric study.

We report the case of an 18-year-old woman who had a small-cell malignant neoplasm of the left paranasal sinuses and who, 18 months later, developed malignant ascites. Immunoperoxidase stains of the ascitic fluid cells and, subsequently, the original tumor, identified it as embryonal rhabdomyosarcoma. Flow cytometric DNA analysis of the ascitic fluid demonstrated an aneuploid cell population that was also present in the paraffin-embedded tissue on which the original diagnosis had been made.

The vacuolated glycogen-laden leukemic lymphocytes of a T-cell lymphoproliferative disorder.

In this report, we present a case of peripheral T-cell leukemia/lymphoma having mostly small- to medium-sized cells with abundant clear cytoplasmic vacuoles. The presentation at the time of diagnosis was one of leukemia/lymphoma. The phenotype of the leukemic cells of the peripheral blood was T1+, T11+, TQ1+, interleukin-2+, T3-, T4-, and T8-. The cells in the peripheral blood as well as those obtained from lymph node biopsy were strongly periodic acid-Schiff positive; the positivity was diastase sensitive. Electron microscopy confirmed the presence of glycogen in the cytoplasmic vacuoles. Serologic tests were negative for human T-cell lymphotropic virus type 1 antibody. The data presented in this article support the existence of a vacuolated variant of peripheral T-cell leukemia/lymphoma and further expand the morphological spectrum of T-cell lymphoproliferative disorders.

Differential expression of c-myc and H-ras oncogenes in Barrett's epithelium. A study using colorimetric in situ hybridization.

To determine the role of c-myc and H-ras in progressive, dysplastic Barrett's mucosa (BM), and the usefulness of these oncogenes as markers for dysplastic lesions at high risk for malignant transformation, sequential formaldehyde solution-fixed, paraffin-embedded biopsy specimens that were obtained from 12 patients with BM were evaluated by in situ hybridization with the use of biotinylated complimentary DNA probes. Nine of the patients were taken from a previous prospective study. Four of these nine patients had dysplasia, and adenocarcinoma had developed in two of them; five had nondysplastic BM only. Two additional patients had adenocarcinoma, but their initial biopsy specimens had revealed dysplasia. One additional patient had intermediate-grade dysplasia. The intensity of oncogene expression was quantified by computerized color-image analysis. Enhanced c-myc expression of approximately equal intensity was consistently observed in all grades of dysplasia and carcinoma. H-ras was also consistently expressed in higher grades of dysplasia and carcinoma but not in low-grade dysplasia. Neither c-myc nor H-ras expression was detected in nondysplastic BM. The expression of H-ras in dysplastic BM appears to be a helpful marker for identifying which dysplastic lesions will progress to carcinoma.

Possible selective elimination of red blood cells under the influence of colloidal silica.

The physical interaction between the red blood cells and colloidal silica leads to hemolysis. We propose that the nature of the red-cell elimination as the result of this interaction is not random. As the negatively charged silica particles at the physiological conditions electrostatically react with the positive sites of the outer side of the cell membrane, it seems reasonable to suppose that the cells with a low density of negative charges on the membrane surface area are more probable to be lysed in the first place.

Salivary gland neoplasms with basaloid cell features: report of two cases diagnosed by fine-needle aspiration cytology.

Basal-cell adenoma and basal-cell adenocarcinoma of the salivary gland are rare tumors. Fine-needle aspiration cytology of these tumors, particularly those of basal-cell adenocarcinoma, has rarely been described in the literature. In this report, we describe the clinical, cytomorphologic, histopathologic, and immunohistochemical features of basal-cell adenoma and its malignant counterpart, basal-cell adenocarcinoma, in 2 patients. Fine-needle aspiration specimens from both tumors contained abundant cohesive groups of neoplastic cells. Basaloid cells were prominent in both tumors; however, there were significant cytologic atypia, hyperchromasia, and increased nuclear-to-cytoplasmic ratio in basal-cell adenocarcinoma. Review of the literature and cytomorphologic distinction between both tumors and others are discussed.

Flow cytometric DNA analysis.

Flow cytometric DNA analysis, including principles, techniques, and applications, is reviewed. Flow cytometry, a relatively recently developed technology, is being increasingly used in the diagnosis and treatment of a wide variety of benign and malignant diseases. Current major applications include phenotypic analysis of cells, cell sorting, and DNA analysis. DNA analysis by flow cytometry is rapid, reliable, and reproducible. Flow cytometric DNA analysis offers a quick, reliable method of analyzing cellular DNA content capable of identifying cell populations with abnormalities in total DNA content.