[3H]Ro 15-1788 binding sites to brain membrane of the saltwater Mugil cephalus.

The equilibrium binding parameters of the benzodiazepine antagonist [3H]Ro 15-1788 (8-fluoro-3-carboethoxy-5,6-dihydro-5-methyl-6-oxo-4H-imidazol-[1,5-a]-1,4 benzodiazepine) were evaluated in brain membranes of the saltwater teleost fish, Mugil cephalus. To test receptor subtype specificity, displacement studies were carried out by competitive binding of [3H]Ro 15-1788 against six benzodiazepine receptor ligands, flunitrazepam [5-(2-fluoro-phenyl)-1,3-dihydro-1-methyl-7-nitro-2H-1,4-benzodiazepin-2-one], alpidem [N,N-dipropyl-6-chloro-2-(4-chlorophenyl)imidazo[1,2-a]pyridine-3-acetamide], zolpidem [N,N-6 trimethyl-2-(4-methyl-phenyl)imidazo[1,2-a]pyridine-3-acetamide hemitartrate], and beta-CCM (methyl beta-carboline-3-carboxylate). Saturation studies showed that [3H]Ro 15-1788 bound saturatably, reversibly and with a high affinity to a single class of binding sites (Kd value of 1.18-1.5 nM and Bmax values of 124-1671 fmol/mg of protein, depending on brain regions). The highest concentration of benzodiazepine recognition sites labeled with [3H]Ro 15-1788 was present in the optic lobe and the olfactory bulb and the lowest concentration was found in the medulla oblongata, cerebellum and spinal cord. The rank order of displacement efficacy of unlabelled ligands observed suggested that central-type benzodiazepine receptors are present in one class of binding sites (Type I-like) in brain membranes of Mugil cephalus. Moreover, the uptake of 36Cl- into M. cephalus brain membrane vesicles was only marginally stimulated by concentrations of GABA that significantly enhanced the 36Cl- uptake into mammalian brain membrane vesicles. The results may indicate a different functional activity of the GABA-coupled chloride ionophore in the fish brain as compared with the mammalian brain.

Autoradiographic distribution of peripheral benzodiazepine receptors in the retina of the albino rabbit, Lepus cunicula.

The distribution of peripheral benzodiazepine receptors (PBRs) in the retina of the albino rabbit, Lepus cunicula, was studied by autoradiography using [3H]-PK11195, a isoquinoline carboxamide, as a tracer. Autoradiograms obtained by directly placing the slides containing the retina sections on tritium-sensitive film provide evidence for the presence of PBRs in rabbit retina. Furthermore, the dark field examination of photomicrographs taken from autoradiograms showed two dense horizontal bands corresponding to the outer and inner photoreceptor segments, and to the inner plexiform layer. The retinal regions where [3H]-PK11195 binding was more dense are rich in mitochondria, suggesting that as in other neuronal tissues, retinal PBRs are involved in the mitochondrial activity.

Multiple regions of chromosome 6q affected by loss of heterozygosity in primary human breast carcinomas.

A total of 80 primary human breast carcinoma DNAs were analysed for loss of heterozygosity (LOH) on the long arm of chromosome 6, using microsatellite markers whose location has been defined physically and by linkage analysis. Loss of heterozygosity was observed in 38 of 80 (48%) tumours that were informative for at least one locus. The analysis revealed partial or interstitial deletions of chromosome 6q. Detailed mapping of chromosome 6q in these tumour DNAs identified two and perhaps three commonly deleted regions. One of these is located between markers D6S251 and D6S252 (6q14-q16.2), another between D6S268 and D6S261 (6q16.3-q23) and a third between D6S287 and D6S270 (6q22.3-q23.1).

Detection of DNA mutations in acid formalin-fixed paraffin-embedded archival tumor specimens by polymerase chain reaction-single strand conformation polymorphism analysis.

A single strand conformation polymorphism analysis on polymerase chain reaction (PCR)-amplified genomic DNA was used for the detection of DNA mutations in acid formalin-fixed, paraffin-embedded tissues. Fifty non-small cell lung cancers were screened for mutations in the exon 5 of the p53 tumor suppressor gene. Structural abnormalities of the gene were found in nine (18%) of the tumors examined. The results show that paraffin-embedded tissues fixed in unbuffered formalin can be a good source of DNA for a mobility shift analysis by neutral polyacrylamide gel electrophoresis. The described procedure allows retrospective genetic studies on paraffin blocks available in all pathology departments.

P53 and C-erbb-2 alterations in in-situ and invasive ductal breast carcinomas - a genetic and immunohistochemical analysis.

p53 mutations, c-erbB-2 amplifications and expression of the related proteins were evaluated in a panel of ductal breast carcinomas selected on the basis of their invasive component. The tumors comprised: 8 ductal carcinomas in situ (DCIS); 8 carcinomas with a minimal (less than 20%) invasive component, hereafter referred to as DCIC (<20%); 13 carcinomas with 20%-50% invasiveness, DCIC (20-50%), and 48 infiltrating carcinomas with more than 50% invasive component, DCIC (>50%). Tumors were further subdivided into large pleomorphic cell type or small regular cell type. A strong association was present between p53 gene mutations and p53 protein overexpression (p<0.001) as well as between amplification of the c-erbB-2 gene and expression of its protein product (p=0.006). p53 aberrations (gene mutation and/or protein overexpression) were observed in 1 (12%) of 8 DCIS, 1 (11%) of 9 DCIC (<20%), 3 (23%) of 13 DCIC (20%-50%), and 13 (28%) of 47 DCIC (>50%). Amplification and/or overexpression of c-erbB-2 were found in 30 (39%) of the 77 breast carcinomas analyzed and were more frequent in DCIC (<20%) and in DCIC (20%-50%) (56% and 46% respectively) than in DCIS or DCIC (>50%) (12% and 38% respectively). Irrespective of the presence of invasion, tumors with p53 or c-erbB-2 alterations showed more frequently large cells with pleomorphic nuclei, (for p53, p=0.027; for c-erbB-2, p=0.014). Our data suggest that p53 and c-erbB-2 alerations may occur in the earliest recognized phase of breast cancer and may be important in the evolution of small cell to large cell mammary carcinoma during tumor progression.

p53 mutations and histological type of invasive breast carcinoma.

A polymerase chain reaction-single strand conformation polymorphism assay was used to assess p53 mutations in 148 invasive breast carcinomas, selected on the basis of their histotype. They comprised 56 lobular, 47 ductal, 19 mucinous, 18 medullary, and 8 papillary carcinomas. The distribution of p53 mutations was significantly different (P = 0.006) in the histotypes examined: mutations were frequent in medullary (39%) and ductal (26%), less common in lobular (12%), and absent in mucinous and papillary carcinomas. The frequency of mutations in the exon 5 of the p53 gene was significantly higher in medullary carcinomas than in the other histotypes: 5 (63%) of the mutations found in exon 5 were observed in medullary carcinomas (P = 0.012). One hundred twenty-two tumors from the total were also examined by immunohistochemistry for p53 overexpression using antibody PAb 1801. A specific immunostaining in neoplastic cells was present in 12 tumors. A strong correlation (P < 0.001) was observed between p53 mutations and nuclear accumulation of the p53 protein: 10 tumors were scored positive for both p53 mutation and overexpression. However, in 9 cases having a mutated p53 gene we failed to find a positive immunoreaction. A significant association (P = 0.01) was present between mutations in the p53 gene and high proliferative activity of the tumors determined by immunohistochemistry with monoclonal antibody Ki-67. Moreover, a significantly higher expression of the Ki-67 antigen was found in medullary carcinomas compared to the other histotypes. Our findings indicate that in invasive breast carcinomas structural abnormalities of the p53 gene are mainly seen in medullary and ductal tumors and that the other histological types, especially those associated with a high level of differentiation and favorable prognosis, show a very low incidence of p53 mutations.

Interferon-alpha/beta in virus-induced mouse mammary carcinogenesis: effects on the spontaneous process and on the progression of transplanted pre-neoplastic lesions.

Low levels of anti-viral activity, mainly interferon alpha/beta (IFN-alpha/beta), are regularly found in lymphoid tissues of BALB/c mice infected with the C3H strain of mammary tumor virus. At the time of tumor development, significant amounts of anti-viral activity were detected in homogenates of spleen and mammary tumors, but not in blood and normal mammary glands. This activity is pH2-resistant and neutralized by antibody to IFN/alpha-beta. The pathogenetic role of IFN in mammary carcinogenesis was investigated in 2 ways: (a) by treating virus-infected newborn mice with antibody to IFN-alpha/beta, and (b) by giving either the latter antibody or IFN-alpha/beta to virus-free animals transplanted with pre-neoplastic lesions. Mice were treated only for 2 months, starting either 1 week after birth or immediately after tumor transplant. In case (a), treatment with antibody to IFN-alpha/beta shortened the incubation period of mammary carcinomas and decreased the mean survival time. In case (b), anti-IFN antibody did not significantly affect the development of mammary tumors. However, exogenous IFN-alpha/beta markedly reduced both tumor incidence and mortality rate. These results indicate that endogenous IFN-alpha/beta plays a crucial role in the in vivo restriction of the early infectious phase of spontaneous carcinogenesis and that relatively high doses of IFN-alpha/beta may inhibit the progression of pre-neoplastic lesions.

Decreasing expression of NM23 gene in metastatic murine mammary tumors of viral etiology (MMTV).

Murine mammary tumors induced by the Murine Mammary Tumor Virus (MMTV) were chosen to study the expression of the NM23 gene during the metastatic process because of their viral etiology, different from that of the previously reported experimental tumor systems. NM23 mRNA levels are higher in non metastatic tumors than in metastatic ones. Moreover, the NM23 expression is higher in tumors induced by the C3H variant of the MMTV than in tumors induced by the RIII variant. These data are a further support to the hypothesis of a basic role of the NM23 gene in the down-regulation of tumor progression.

Host genetic background effect on the frequency of mouse mammary tumor virus-induced rearrangements of the int-1 and int-2 loci in mouse mammary tumors.

The frequency with which int-1 and int-2 are rearranged in mouse mammary tumors by mouse mammary tumor virus (MMTV)-induced insertional mutagenesis is a consequence of the host genetic background. In 75% of C3H mammary tumors, int-1 is rearranged by MMTV insertion, whereas only 30% of BALB/cfC3H tumors contain a virus-induced rearrangement of int-1. This difference is significant (P less than 0.005) and could not be accounted for by the potentially additive effect of the genetically transmitted Mtv-1-encoded virus in C3H mice. Similarly, MMTV-induced rearrangement of the int-2 gene in mammary tumors of the R111 mouse strain (59%) occurred at a significantly (P less than 0.025) higher frequency than in BALB/cfR111 (25%) mammary tumors. Moreover, in BALB/cfR111 mammary tumors, there is evidence that rearrangement of int-1 and int-2 does not occur independently (P less than 0.025). These results suggest that the long history of inbreeding for high tumor incidence of C3H and R111 mouse strains has selected for the fixation of host mutations which either complement the action of the particular int gene or affect the sensitivity of specific subpopulations of mammary epithelium to infection by particular strains of MMTV.

Silver enhancement of protein A-gold probes on resin-embedded ultrathin sections. An electron microscopic localization of mouse mammary tumor virus (MMTV) antigens.

A simple method is described allowing the enhancement of the visibility of small gold probes for the electron microscopy. This method, which allows the silver intensification of gold directly on epon-embedded ultrathin sections, was used for the electron microscopic localization of Mouse Mammary Tumor Virus (MMTV) antigens in cultured cells derived from GR and BALB/cfRIII mouse mammary tumors. After the immunostaining with the preembedding protein A-gold technique, the ultrathin sections, placed on 200 mesh copper grids, were rehydrated and exposed to a photographic developer containing silver nitrate. During this physical development gold particles are incapsulated in growing shells of metallic silver, which gradually become more and more visible. We were able to obtain a heavy labelling of the viral particles, well visible even at low magnification, with a negligeable background staining. The present technique can be useful whenever it is necessary to use the smallest gold probes today available.

Reciprocal interference between milk-transmitted mammary tumor virus and Friend leukemia viruses in mice: possible role of the interferon system.

We have previously shown that mice simultaneously infected with the murine mammary tumor virus (MuMTV) and with certain slow murine leukemia viruses (MLV) have an increased resistance to the pathological effects of both agents. Here we report that milk-transmitted MuMTV also delays the development of the acute erythroleukemia induced by Friend leukemia virus (FLV), and retards, or prevents in some cases, the development of long-term lymphomas caused by its helper component. This is confirmed by the observation that the average life span of MuMTV-carrying mice infected with FLV or its helper component is prolonged by over 30% as compared to that of MuMTV-free animals infected with the same agents and by the finding that the replication of Friend viruses is reduced in mice neonatally exposed to MuMTV. Since the antibody responses of mice to MuMTV and to FLV were not cross-reactive, we searched for antiviral activity in the tissues of mice exposed to MuMTV, FLV, or the helper component of FLV. Low levels of interferon-alpha/beta were consistently detected in spleen extracts from mice infected with all these agents but could not be demonstrated in the spleens of uninfected BALB/c mice; thus, the chronic production of endogenous interferon is likely to play a major role in the reciprocal interference in vivo between retroviruses belonging to different genera.

Lobuloalveolar differentiation and tumorigenesis: two separate activities of mouse mammary tumor virus.

The morphological growth of the mammary glandular tree from 3 to 18 months of age has been quantified in three groups of genetically identical virgin female mice: BALB/c mice free of milk-transmitted mouse mammary tumor virus (MMTV) infection and BALB/cfC3H and BALB/cfRIII mice carrying milk-transmitted MMTV infection of C3H or RIII origin, respectively. Mice were killed at 3-month intervals, and their mammary glands were prepared for stereoscopic examination as whole mounts. Any cluster of alveoli from 0.1 mm in diameter was counted and measured at 0.1-mm intervals. The small clusters of alveoli (less than or equal to 0.3 mm) were used to measure the amount of lobuloalveolar differentiation. The larger clusters of alveoli, the hyperplastic alveolar nodules, and the mammary tumors were used to measure the frequency of tumor cell transformations. Lobuloalveolar differentiation in BALB/c mice was increased significantly by both C3H (p less than 0.001) and RIII (p less than 0.001) MMTV infections. RIII MMTV induced a significantly higher lobuloalveolar differentiation as compared to that induced by C3H MMTV (p less than 0.001). C3H MMTV showed a significantly higher capability in inducing hyperplastic alveolar nodules and mammary tumors as compared to that of RIII MMTV (p less than 0.001). The conclusions were that two different activities are carried out by MMTV, namely, lobuloalveolar differentiation and noduligenesis, and that these two MMTV activities are distinct from each other.

Development, morphology, and progression of mammary tumors during and after fertile life in BALB/cfRIII mice.

A study was done on 368 BALB/cfRIII mammary tumor-bearing breeding female mice that are of the BALB/c genotype and carry milk-transmitted RIII murine mammary tumor virus (MuMTV) infection initiated by foster-nursing. All tumors were characterized by chronologic, biologic, and morphologic analyses. Mammary tumors occurring during pregnancy-lactation periods were mostly pregnancy-dependent plaques showing organoid tubular structure, sudden growth during late pregnancy, regression after delivery, and slow progression. The frequency of pregnancy-dependent tumors per 100 mice at risk increased steadily from first (1 tumor) to sixth (38 tumors) pregnancy-lactation period. Mammary tumors occurring after fertile life were mostly characterized by an irregular, slow growth and a type B morphology. The frequency of these tumors per 100 mice at risk per 100 days of survival also increased regularly in females undergoing from one (73 tumors) to six (117 tumors) pregnancy-lactation periods. Data were discussed in the light of the pattern of MuMTV release through BALB/cfRIII milk.

Quantitative determination of mammary tumor virus in individual samples of mouse milk.

The possibility to determine quantitatively the intact mammary tumor virus (MTV) in milk of mice carrying milk-transmitted MTV has been assayed by a method that allows direct comparison between individual milk samples. The method is based on (a) the measure of light scattering of partially purified MTV preparations, (b) the use of milk from genetically identical MTV free mice as blank and (c) the quantitative reference to the total protein content of whole milk. The sensitivity, specificity and reproducibility of the procedure, as well as the requirement of appropriate quantitative references, are illustrated and discussed. BALB/c (MTV free), BALB/cfC3H, and BALB/cfRIII mice have been used.

Quantitative comparison of milk-released C3H and RIII mammary tumor viruses in infected BALB/c hosts.

The C3H and RIII mammary tumor viruses (MTV) carried by BALB/cfC3H and BALB/cfRIII breeding females have been quantified and compared in milk samples, after partial purification with a sucrose density gradient. The samples were collected at identical times during the first 3 lactation periods from individual mice (6 per strain), standardized for age at delivery and size of litter. Milk samples from 6 MTV negative BALB/c controls have also been analyzed. Data for comparison are expressed in optical density units (ODU) and refer to the protein content of the whole milk using MTV-negative Balb/c milk as blank. The results have shown 1) an increase of MTV released through milk in each MTV-carrying female from the first (average ODU, 0.542) to the second (1,351) and third (2.105) lactation, 2) individual variations, and 3) a significant difference in release between C3H and RIII MTV, the latter being more than double (average ODU, 1.801) in respect to the former (0.847). The apparent discrepancy between these results and the bioactivity in BALB/c mice of C3H and RIII MTV, significantly lower for the latter, is discussed.

Bioactivity of C3H and RIII mammary tumor viruses in virgin female BALB/c mice.

The bioactivities of C3H and RIII mammary tumor virus (MuMTV) in virgin female BALB/c mice differed. The average number of mammary hyperplastic alveolar nodules per mouse after noduligenic tests was 20.1 in BALB/cfC3H and 10.7 in BALB/cfRIII females. Spontaneous mammary tumor incidence after 20 months of observation was 47.5% in BALB/cfC3H and 14.6% in BALB/cfRIII females (P less than 0.01). The frequency of lung metastases in mammary tumor-bearing mice was 63.1% in BALB/cfC3H and 16.6% in BALB/cfRIII females (P less than 0.05), although the clinical duration of mammary tumors was the same. These data demonstrated a lower bioactivity of RIII MuMTV when compared to C3H MuMTV in BALB/c mice and suggested that the causative virus may control all the steps of mouse mammary tumor development, including metastasis.