Evidence that mirex promotes a unique population of epidermal cells that cannot be distinguished by their mutant Ha-ras genotype.

Mirex is a potent tumor promoter in 7,1 2-dimethylbenz[a]anthracene (DMBA)-initiated female CD-1 mouse skin. Like 12-O-tetradecanoylphorbol-13-acetate (TPA), mirex promotes papillomas that have a Ha-ras mutation; however, unlike TPA promotion, mirex promotion does not involve a general hyperplastic response. We used proliferating cell nuclear antigen (PCNA) and 5-bromo-2'-deoxyuridine (BrdU) immunohistochemical staining to further examine the proliferative capacity of mirex. The numbers of PCNA- and BrdU-positive epidermal S-phase cells were highly concordant in all treatment groups. Unlike a single application of TPA, a single application of mirex had little or no effect on the number of S-phase epidermal cells, and chronic application of mirex to mouse skin produced only minimal increases in S-phase cells. Moreover, mirex did not significantly alter the growth of BALB/MK-2 keratinocytes in media containing either 0.05 or 1.2 mM Ca++. These results suggest that mirex may have highly specific effects on the proliferation of initiated cells and support the existence of a unique mirex mechanism and/or distinct population of mirex-promotable mutant Ha-ras epidermal cells. To begin to address this issue of a distinct population of mirex-promotable mutant Ha-ras cells, we conducted a tandem experiment in which DMBA-initiated mice were treated twice weekly with a maximal promoting dose of mirex. Then, when the number of papillomas reached a plateau, these same mice were treated twice weekly with a maximal promoting dose of TPA. Mice treated with mirex developed a maximum of 6.4 papillomas/mouse. These mice were then promoted with TPA, which produced 8.9 additional papillomas/mouse for a total of 15.3 papillomas/mouse. The maximum tumor yields from other groups of mice treated with only TPA or mirex were 9.8 and 7.3 papillomas/mouse, respectively. Therefore, under these tandem conditions, tumor yields were additive, indicating that there are at least two distinct populations of mutant Ha-ras cells: one promoted by mirex and the other by TPA.

Immunohistochemical expression of SKALP/elafin in squamous cell carcinoma of the oesophagus.

In this study, the immunohistochemical expression of a new inducible elastase inhibitor, SKALP (skin-derived anti-leucoproteinase)/elafin, in the tissue of squamous cell carcinoma and uninvolved oesophageal mucosa was studied using a polyclonal rabbit anti-serum against SKALP/elafin. The results were compared with the immunohistochemical staining of proliferating cell nuclear antigen (PCNA) and the TUNEL assay in serial sections. In non-malignant oesophageal mucosa, the expression of SKALP/elafin was localized in the cells of the stratified zone overlying the PCNA-positive basal zone. In oesophageal cancer, the incidence of the expression was significantly related to the degree of the differentiation of the tumour. Characteristically, the expression was almost limited in tumour cell nests that had a clear squamous phenotype. In tumour cell nests, the expression of SKALP/elafin was localized in the cells overlying PCNA-expressing cells and no expression was found in the cells that expressed PCNA; DNA fragmentation was often observed in the same cell layers as those in which SKALP/elafin immunoreactivity was found. This enzyme inhibitor is speculated to be involved in the induction of the cell differentiation and apoptosis of human squamous cell carcinoma cells of the oesophagus.

Different carcinogenesis in the gastric remnant after gastrectomy for gastric cancer.

BACKGROUND: The incidence of gastric remnant cancer after surgery for gastric malignancies has been increasing. The interval between previous operations and the diagnosis of gastric remnant cancer, location of cancer development, and histologic type were different from those after surgery for benign diseases. However, very little is known about the reasons for these differences. Patients with gastric cancer already have cancer-related gastric mucosal changes at gastrectomy, and they undergo a wide range of dissection of nerve distribution to the stomach due to lymph node dissection. Therefore, the effects of preliminary administration of a carcinogenic agent and denervation of the gastric mucosa on tumorigenesis in the gastric remnant were investigated. METHODS: Using male Wistar rats, N-methyl-N'-nitro-N-nitroguanidine (MNNG; 50 mg/L) was given in drinking water for 10 weeks. The animals were then assigned into four groups of those undergoing Billroth I (B-I) gastrectomy or Billroth II (B-II) gastrectomy, with and without denervation. Subdiaphragmatic truncal vagotomy was performed in the denervated group. Thirty weeks after gastrectomy, the following investigations were performed: histologic examination and periodic acid-Schiff-Alcian blue (PAS-AB) staining of the gastric mucosa by immunohistochemistry of proliferating cell nuclear antigen (PCNA). RESULTS: In macroscopic findings, the groups undergoing nitrosoguanide (NG) gastrectomy with denervation showed a significant increase in the development of whitish, nodular changes in the gastric body. These changes mainly consisted of intestinal metaplasia in microscopic findings. In the NG gastrectomy group, the cancer developed at a lower rate of incidence at the anastomotic site and in the gastric body (1 of 11 rats and 1 of 11 rats, respectively). Conversely, a higher incidence of cancer development (5 of 13 rats) in the gastric body was observed in the group that underwent NG gastrectomy with denervation. Furthermore, the denervation group showed a significant increase in the PCNA labeling index and a distinct increase in the staining of Alcian blue positive mucin in the mucosa of the gastric body. The cancers that developed in the gastric body showed horizontal growth and were accompanied by intestinal metaplasia. In contrast, the cancers that developed in the gastric stumps showed downward growth, and were always accompanied by adenocystic proliferation, but not intestinal metaplasia. CONCLUSIONS: Different processes of carcinogenesis in the gastric remnant are postulated after the surgery for gastric malignancies. The developed cancer is defined by its location and the gastric mucosal changes that developed at the time of gastrectomy.

Malignant inverted papilloma of the urinary bladder: the histopathological aspect of malignant potential of inverted papilloma.

To investigate the histopathological characteristics of inverted papillomas of the urinary bladder, including the possibility of malignant transformation, we studied the indicators of cellular proliferation activity in 7 inverted papillomas of the bladder including two cases of malignant inverted papilloma of the bladder. PCNA expression rates in two cases of malignant inverted papilloma were higher than in benign inverted papillomas. Mean numbers of AgNORs per nucleus in malignant inverted papillomas were much more than in benign inverted papillomas. The c-erbB-2 oncoprotein was expressed only in malignant inverted papillomas. These results suggest that PCNA expression rate, mean number of AgNORs per nucleus and c-erB-2 oncoprotein expression may be merited as good indicators to detect the inverted papilloma with more proliferative and aggressive lesions, and with the potential of malignant transformation.

Malignant transformation of the human endometrium is associated with overexpression of lactoferrin messenger RNA and protein.

In the mouse uterus, lactoferrin is a major estrogen-inducible uterine secretory protein, and its expression correlates directly with the period of peak epithelial cell proliferation. In this study, we examine the expression of lactoferrin mRNA and protein in human endometrium, endometrial hyperplasias, and adenocarcinomas using immunohistochemistry, Western immunoblotting, and Northern and in situ RNA hybridization techniques. Our results reveal that lactoferrin is expressed in normal cycling endometrium by a restricted number of glandular epithelial cells located deep in the zona basalis. Two thirds (8 of 12) of the endometrial adenocarcinomas examined overexpress lactoferrin. This tumor-associated increase in lactoferrin expression includes an elevation in the mRNA and protein of individual cells and an increase in the number of cells expressing the protein. In comparison, only 1 of the 10 endometrial hyperplasia specimens examined demonstrates an increase in lactoferrin. We also observe distinct cytoplasmic and nuclear immunostaining patterns under different fixation conditions in both normal and malignant epithelial cells, similar to those previously reported in the mouse reproductive tract. Serial sections of malignant specimens show a good correlation between the localization of lactoferrin mRNA and protein in individual epithelial cells by in situ RNA hybridization and immunohistochemistry. Although the degree of lactoferrin expression in the adenocarcinomas did not correlate with the tumor stage, grade, or depth of invasion in these 12 patients, there was a striking inverse correlation between the presence of progesterone receptors and lactoferrin in all 8 lactoferrin-positive adenocarcinomas. In summary, lactoferrin is expressed in a region of normal endometrium known as the zona basalis which is not shed with menstruation and is frequently overexpressed by progesterone receptor-negative cells in endometrial adenocarcinomas.

Malignant inverted papilloma of the urinary bladder: the histopathological aspect of malignant potential of inverted papilloma

To investigate the histopathological characteristics of inverted papillomas of the urinary bladder, including the possibility of malignant transformation, we studied the indicators of cellular proliferation activity in 7 inverted papillomas of the bladder including two cases of malignant inverted papilloma of the bladder. PCNA expression rates in two cases of malignant inverted papilloma were higher than in benign inverted papillomas. Mean numbers of AgNORs per nucleus in malignant inverted papillomas were much more than in benign inverted papillomas. The c-erbB-2 oncoprotein was expressed only in malignant inverted papillomas. These results suggest that PCNA expression rate, mean number of AgNORs per nucleus and c-erB-2 oncoprotein expression may be merited as good indicators to detect the inverted papilloma with more proliferative and aggressive lesions, and with the potential of malignant transformation.

High-performance liquid chromatographic determination of N-nitroso-N-alkylureas by pre-column fluorescence derivatization and application to blood analysis.

A method for the derivatization and separation of N-nitroso-N-alkylureas [alkyl = methyl (NMU), ethyl (NEU), and n-butyl (NBU)] has been developed. Fluorescent derivatives were formed with sodium sulphide, taurine and o-phthalaldehyde and separated by reversed-phase high-performance liquid chromatography. The limits of detection of standard NMU, NEU and NBU were 0.25, 0.8 and 1.5 pmol/200 microliters, respectively. The method was applied to the determination of NMU in blood after extraction with acetonitrile in the presence of calcium chloride. NBU was used as the internal standard. The recovery of NMU from blood was ca. 95%, and the limit of detection was 10 pmol/400 microliters blood. NMU levels in rabbit blood following a single oral administration were also measured.

Trace analysis of nitrosated foodstuffs for nitrosamides.

An analytical procedure previously developed for the trace determination of nitrosamides was applied to a screening of nitrosated foodstuffs for nitrosoureas. Different types of foodstuffs were nitrosated both under chemical conditions using a high nitrite concentration, and under simulated gastric conditions. Methylating activity corresponding to N-nitroso-N-methylurea (MNU) was detected in most samples. Under chemical conditions, the yields spanned several orders of magnitude with processed fish and meat products being at the top, and plant products at the bottom of the scale. After nitrosation under simulated gastric conditions, the range of MNU activity was significantly smaller. No correlation exists between the yields determined under chemical and simulated gastric conditions.

Trace analysis of N-nitrosoureas by their alkylating activity.

N-Nitrosoureas can be analysed indirectly by gas chromatography with chemoluminescence detection after reaction of the corresponding diazoalkanes with the scavenger reagent N-nitroso-N-tert-butylglycine. A lower determination limit of 2 ng N-methyl-N-nitrosourea per sample is achieved. The method is specific for N-nitroso compounds, which release nonpolar diazoalkanes upon alkaline treatment.

Chemical stability of ecomustine, a new antitumor agent in aqueous and biological media as assessed by high-performance liquid chromatography.

Ecomustine, or CY233 (NSC-609224), is a new water-soluble nitrosoureido sugar derived from acosamine. A high-performance liquid chromatographic assay (HPLC) developed to quantify the unchanged drug in aqueous solutions and biological specimens enabled us to study the chemical stability as a function of pH, light, and temperature. In buffered aqueous solutions, the kinetics of degradation of CY233 is a first-order process. The log k-pH profile demonstrated hydroxide ion-catalyzed solvolysis. The drug is most stable at pH 4, more stable than some other nitrosoureas in 5% glucose (t1/2, 62-67 h) and in 0.9% isotonic saline (t1/2, 25-37 h) solutions. Based on these findings, blood samples should be collected in cold tubes (4 degrees C) containing citrate buffer (pH 4) and all manipulations should be protected from heat and light.

Degradation and disposal of some antineoplastic drugs.

Bulk quantities and pharmaceutical preparations of the antineoplastic drugs carmustine (BCNU), lomustine (CCNU), chlorozotocin, N-[2-chloroethyl]-N'-[2,6-dioxo-3-piperidinyl]-N-nitrosourea (PCNU), methyl CCNU, mechlorethamine, melphalan, chlorambucil, cyclophosphamide, ifosfamide, uracil mustard, and spiromustine may be degraded using nickel-aluminum alloy in KOH solution. The drugs are completely destroyed and only nonmutagenic reaction mixtures are produced. Destruction of cyclophosphamide in tablets requires refluxing in HCl before the nickel-aluminum alloy reduction. Streptozotocin, chlorambucil, and mechlorethamine may be degraded using an excess of saturated sodium bicarbonate solution. The nitrosourea drugs BCNU, CCNU, chlorozotocin, PCNU, methyl CCNU, and streptozotocin were also degraded using hydrogen bromide in glacial acetic acid. The drugs were completely destroyed but some of the reaction mixtures were mutagenic and the products were found to be, in some instances, the corresponding mutagenic, denitrosated compounds.

Decomposition of N-(2-chloroethyl)-N-nitrosocarbamoyl amino acid amides.

The chemical decomposition of N-(2-chloroethyl)-N-nitrosocarbamoyl (Q(NO] prolinamide and valinamide were studied under physiological conditions. The volatile products were identified with GC. Q(NO)-Pro-NH2 gave twice the amount of ethylene glycol and only one-fifth of the 2-chloroethanol produced by Q(NO)-Val-NH2 or BCNU, pointing to different pathways of their decomposition. The carbamoylating activity was also investigated in the presence of cyclohexylamine, and it was found to lead mainly to intramolecular carbamoylation with the formation of hydantoin derivatives.

DNA-nitrosourea interactions. High-performance liquid chromatography of cross-linked dinucleosides and substituted deoxynucleosides.

A reconstituted mixture of five cross-linked dinucleosides possibly involved in DNA-nitrosourea interactions, and of their degradation products (nucleobases, deoxynucleosides and mono- or disubstituted deoxynucleosides), was analysed by reversed-phase high-performance liquid chromatography using C18 columns and an diode-array detector. The chromatographic conditions for separating the twenty-one investigated compounds were optimized, and the compounds were identified by both their retention times and their UV spectra. A structure-retention time relationship was observed under suitable conditions and is discussed. Its validity was confirmed by the prediction of the retention time of a new cross-linked dinucleoside synthesized for this purpose.

[ACNU delivery to malignant glioma tissue by osmotic blood brain barrier modification with intracarotid infusion of hyperosmoral mannitol].

Drug delivery to the tumor has been one of the major subjects in the field of brain tumor chemotherapy because of blood brain barrier. Recent studies including quantitative autoradiographic studies revealed that blood brain barrier is present and intact in the brain adjacent to tumor where viable tumor cells are infiltrating, and also in the tumors which are early in the development. In 1972 Rapoport et al demonstrated that it is possible to transiently and reversibly open the blood brain barrier by an intracarotid infusion of a hyperosmoral solution. This technique is found to increase cerebrovascular permeability to chemotherapeutic agents. Six cases of glioma, including 4 astrocytoma grade 4, 1 astrocytoma grade 3, 1 astrocytoma grade 2, were treated during operation with intracarotid infusion of ACNU 100 mg/body/5 min. (1.3-2.2 mg/kg) following intracarotid infusion of 20% mannitol 200 ml (1.3-1.6 ml/sec) through the catheter in the internal carotid artery set preoperatively, and ACNU concentration in tumor tissues and blood were measured at 5, 10, 15, 20, 25, 30, 40, 60 minutes after that. On every case mannitol contrast enhancement CT was studied by the intracarotid infusion of 60% conray 100 ml/5 min. following the intracarotid infusion of 20% mannitol 200 ml comparing with contrast enhancement CT and plain CT. Maximum ACNU concentrations in blood were 2.12-4.12 micrograms/ml (mean 3.1 +/- 0.74) at 5 min. after the intraarterial administration of mannitol and ACNU on every case. At 20 min. following the administration ACNU levels were decreased to half level (mean 1.49 +/- 0.42 microgram/ml) and 0.58 +/- 0.18 microgram/ml at 60 min.(ABSTRACT TRUNCATED AT 250 WORDS)

Preparative and analytical high-performance liquid chromatographic methods in the synthesis and analysis of decomposition of nitrosourea nucleosides.

Details are given for reversed-phase, adsorption, and aqueous ammonia-modified adsorption high-performance liquid chromatographic systems developed to separate 3'-chloroethylnitrosourea analogues of thymidine, 2'-deoxyuridine, and 5-fluoro-2'-deoxyuridine from their decomposition products and synthetic precursors. The effect of varying the substituent at the 3'- and 5-position on relative retention in each system is discussed. These systems are used to purify intermediates in the synthesis of these potent antineoplastic agents, and for the simultaneous analysis of the nitrosourea nucleosides and their breakdown products in kinetic studies of their decomposition. Application of these methods to the analysis of the kinetics of the breakdown of these compounds is demonstrated, with detection limits (signal-to-noise ratio = 2) in the 1-2 ng range.

Quantitation of lipophilic chloroethylnitrosourea cancer chemotherapeutic agents.

A simple and rapid quantitative method for the derivatization and determination of lipophilic chloroethylnitrosoureas is described. This procedure involves the ether extraction of the chloroethylnitrosourea from plasma and conversion of the parent drug to an O-methylcarbamate by reaction in anhydrous methanol. The product O-methylcarbamate may be separated with gas chromatography (GC) and detected with nitrogen-specific GC detectors or with mass spectrometry using multiple-ion detection. The lower limit of detection for each method was approximately 100 ng/ml plasma.