Autoradiographic localization of glycoprotein in human breast cancer cells maintained in organ culture after incubation with (3H)fucose or (3H)glucosamine.

Explants of nine infiltrating duct carcinomas of the human female breast, maintained in organ culture, were exposed to the glycoprotein precursors, L-[3H]furcose and [3H]glucosamine, in order to determine the cellular distribution of newly synthesized glycoprotein as revealed by autoradiography with the light and electron microscopes. Explants were incubated with a single isotope for 2 hr, at which time some of the labeled explants were removed for autoradiographic analysis while the rest were transferred to nonradioactive medium for an additional 24 hr. After exposure to label for 24 hr, autoradiography with each isotope was similar and showed strong reactions over most tumor cells. The reactions were due to clumps of silver grains over intracytoplasmic lumina within single tumor cells and silver grains over Golgi saccules, cytoplasmic vesicles, lysosome-like bodies, lateral and basal plasma membranes, and microvilli. Extracellular ductular structures were also heavily labeled. At the later sampling time, Golgi saccules often showed a reduced reaction while the reactions over other organelles and intracellular and extracellular ductular structures remained strong. The observations suggest that in our in vitro system the tumor cells are metabolically active and complete the synthesis of the carbohydrate side chains of glycoproteins within the Golgi apparatus. From there, some of the newly synthesized glycoprotein appears to migrate to plasma membranes and lysosome-like bodies. Furthermore, our data support the notion that many duct carcinomas of the breast exhibit secretory activity by showing that some newly synthesized glycoprotein also appears to become products that are secreted into intracellular and extracellular ductular structures.     

Androgen receptor versus erbB-1 and erbB-2 expression in human prostate neoplasms

A subject of current interest, especially in the development of androgen refractory prostate cancer, is the androgen receptor (AR) activation by growth factor receptors. Here, we report our work on the measurement of AR mRNA and protein expression in benign prostatic hyperplasia (BPH) and prostatic carcinoma (PCA) and evaluation of the relationship between AR, erbB-1 and erbB-2 gene expression determined in the same tissue. In order to define AR, erbB-1 and erbB-2 in human prostate neoplasms 36 benign prostatic hyperplasia, 46 prostatic carcinoma and 12 normal prostate gland samples were analysed. According to distant metastasis PCA tissues were divided into two categories: i) T1-4N0-3M0 (25 samples) and ii) T4N2-3M1 (21 samples). AR, erbB-1 and erbB-2 mRNA expression was estimated by RT-PCR. AR protein expression, both in nuclear and cytoplasmic fractions, was measured by Western blot technique. The association of AR mRNA and protein expression with erbB-1 and erbB-2 gene expression was evaluated. It was found that in clinically invasive (group II of PCA) prostate cancer cases AR mRNA expression was significantly correlated with erbB-2 mRNA expression (Spearman R coefficient 0.86, p<0.05). Interestingly, AR protein expression in this group of PCA was determined mainly in nuclear fraction. By Western blot AR protein was identified in 76.0% (16/21) and 23.8% (5/21) of PCA group II nuclear and cytoplasmic fractions, respectively. Furthermore, the mean AR protein level in nuclear fraction of clinically invasive (group II) PCA (0.82+/-0.04) was significantly higher (p<0.05) as compared to the normal group (0.56+/-0.11). In the case of T4N2-3M1 samples, significant correlation between AR protein level in nuclear fraction and erbB-2 mRNA expression (Spearman R coefficient 0.53, p<0.05) was stated.     

Expression of α3β1 integrin receptor and its ligands in human lung tumors

Increasing experimental evidence demonstrates that malignant transformation is associated with changes in the repertoire of expression of the integrin family of molecules, which mediate cell-matrix and cell-cell interactions. We have analyzed immuno-histochemically and immunochemically the expression of VLA-3 integrin and its known ligands, namely, laminin (LM), fibronectin (FN), collagen type IV (Coll IV), nicein (NIC), and entactin/nidogen (ENT), in lung tumors of various histological types. α₃β₁ was detectable in normal bronchial epithelium and along basement membranes of alveolar walls. In non-small cell lung carcinomas (NSCLC) the integrin was expressed in 82% of the cases, independently of histological type and degree of differentiation of the tumors. On the other hand, only 13% of the small cell lung carcinomas (SCLC) displayed a weak and heterogeneous distribution of the α₃β₁ complex. Our findings were confirmed immunochemically using long-term tumor cell lines. While the expression of both α₃β₁ and ligands LM, FN, Coll IV, and Ent correlated in NSCLC with the presence of basement membranes, FN was the only ligand detectable in the stroma of SCLCs. A selective loss of nicein in basement membranes was demonstrated in NSCLC indicating an impairment of expression of this glycoprotein following malignant transformation. © 1995 Wiley-Liss, Inc.     

Pharmacokinetics of N'-nitrosonornicotine and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone in laboratory animals

The pharmacokinetics of N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) in the Syrian golden hamster, the CD-1 mouse, and the baboon were compared to the pharmacokinetics in the Fischer rat. The formation and biological half-life of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL), the major metabolite of NNK, was also studied in these animal species. The biological half-life of NNN in these 4 animal species ranged from 0.24 h to 3.06 h, that of NNK from 0.21 h to 0.43 h and NNAL from 0.48 h to 2.9 h. The pharmacokinetic data obtained in the baboon suggest that treatment with NNN and NNK causes an enzyme induction which accelerates the rate of elimination of these compounds.     

Partial deletion of chromosome No. 2 in myelocytic leukemias of irradiated C3H/He and RFM mice.

Chromosomes of mouse myelocytic leukemias that developed in 7 irradiated mice, 3 C3H/He males, 1 RFM female, and 3 RFM males were analyzed with chromosome-banding techniques. Chromosomes No. 2 were partially deleted in 6 of the 7 mice. Although the deleted No. 2 chromosomes varied in size in the 6 mice, one common characteristic was noted in all these deletions: A segment lying between a certain band in the region 2C and a band in the region 2E, including the whole region 2D, was missing. Another consistent abnormality was an addition or a loss of the Y-chromosomes in the fraction of cells in all 6 males. In addition to these consistent abnormalities, various chromosomes had structural abnormalities. The RFM female, which did not have the abnormal No. 2 chromosome, had abnormalities in chromosomes No. 3, 4, 11, 12 and 15 and in the X-chromosome. Of the 20 chromosome pairs, only such chromosomes as No. 1, 5, 8, 14, 17, and 19 and the Y-chromosome did not have the structural abnormalities. The possible role of the partial deletion of the No. 2 chromosome was considered in relation to the development of mouse myeloid leukemias.     

Efficacy and safety evaluation of human reovirus type 3 in immunocompetent animals: racine and nonhuman primates.

PURPOSE: Human reovirus type 3 has been proposed to kill cancer cells with an activated Ras signaling pathway. The purpose of this study was to investigate the efficacy of reovirus in immunocompetent glioma animal models and safety/toxicity in immunocompetent animals, including nonhuman primates. EXPERIMENTAL DESIGN: Racine glioma cells 9L and RG2 were implanted s.c. or intracranially in Fisher 344 rats with or without reovirus antibodies, followed by treatment of reovirus. To study whether reovirus kills contralateral tumors in the brain and to determine viral distribution, we established an in situ dual tumor model followed by reovirus intratumoral inoculation only into the ipsilateral tumor. To evaluate neurotoxicity/safety of reovirus, Cynomolgus monkeys and immunocompetent rats were given intracranially with reovirus, and pathological examination and/or behavioral studies were done. Viral shedding and clinical biochemistry were systematically studied in monkeys. RESULTS: Intratumorally given reovirus significantly suppressed the growth of both s.c. and intracranially tumors and significantly prolonged survival. The presence of reovirus-neutralizing antibodies did not abort the reovirus' antitumor effect. Reovirus inhibited glioma growth intracranially in the ipsilateral but not the contralateral tumors; viral load in ipsilateral tumors was 15 to 330-fold higher than the contralateral tumors. No encephalitis or behavioral abnormalities were found in monkeys and rats given reovirus intracranially. No treatment-related clinical biochemistry changes or diffuse histopathological abnormality were found in monkeys inoculated intracranially with Good Manufacturing Practice prepared reovirus. Microscopic changes were confined to the region of viral inoculation and were dose related, suggesting reovirus intracranially was well tolerated in nonhuman primates. CONCLUSIONS: These data show the efficacy and safety of reovirus when it is used in the treatment of gliomas in immunocompetent hosts. Inoculation of reovirus into the brain of nonhuman primates did not produce significant toxicities.     

Expression of phospholipases γ1, β1, and δ1 in primary human colon carcinomas and colon carcinoma cell lines

The levels of expression of phosphoinositide-specific phospholipase Cs (PLCs) were examined in a series of primary human colon carcinomas and in eight colon carcinoma cell lines by using monoclonal antibodies and cDNA probes for PLCγ1, PLCβ1, and PLCδ1. Western and northern blot analyses of PLCγ1 revealed elevated expression of this isozyme at both the protein and mRNA levels in most tumors when compared with paired adjacent normal mucosa samples (in 11 of 13 pairs in the western blots and 8 of 9 pairs in the northern blots). On the other hand, decreased levels of the PLCδ1 protein were seen in most colon carcinomas (12 of 13 paired samples). The levels of PLCβ1 protein were too low to detect possible differences between the carcinoma and normal mucosa samples. Relatively high expression of PLCγ1 was found in almost all of the eight human colon carcinoma cell lines at both the protein and mRNA levels. Only weak expression of PLCβ1 was detected in these cell lines, by both western and northern blot analyses, and PLCδ1 protein was not detected in any of the carcinoma cell lines. These findings provide evidence that colon carcinomas display altered expression of individual isoforms of PLCs and suggest that increased expression of PLCγ1 may play an important role in colon carcinogenesis. © 1995 Wiley-Liss Inc.     

Migration properties of the human ovarian adenocarcinoma cell line IGROV1: Importance of αvβ3 integrins and vitronectin

Cell migration of ovarian tumoral cells is essential for cell dissemination and for invasion of the submesothelial extracellular matrix (ECM). We have conducted a study of the migratory properties of an ovarian adenocarcinoma cell line (IGROV1) by using 2 distinct methods for the evaluation of cell migration. We found that in a short‐term transfilter migration assay, IGROV1 cells migrated toward vitronectin, fibronectin, type IV collagen and laminin in an integrin‐dependent manner. When migration was evaluated in a wound healing assay, the restitution of the wounded area was stimulated solely by added, exogenous vitronectin and was almost totally dependent on αvβ3 integrin function. Moreover, we demonstrated that αvβ3 was localized in focal contacts restricted to the leading edge of migrating cells, whereas vitronectin notably localized with actin stress fibers and cortical actin. On the other hand, several kinase inhibitors were found to impede migration of IGROV1 induced by vitronectin. It thus appears that αvβ3–vitronectin interactions lead to the activation of multiple signaling pathway including activation of protein kinase C, phosphatidyl‐inositol‐3‐phosphate kinase and protein tyrosine kinase. The “αvβ3–vitronectin system” is therefore essential to the migration of human ovarian carcinoma cells.Int. J. Cancer 80:285–294, 1999. © 1999 Wiley‐Liss, Inc.