Preclinical study on gene therapy of cervical carcinoma using adeno-associated virus vectors.

Approximately 90% of cervical carcinomas are causally linked to infections with high-risk human papillomaviruses (HPVs), whose oncogenicity has been assigned to the continued expression of two early genes, E6 and E7. Reversal of the transformed phenotype by inhibiting E6/E7 gene expression therefore provides a suitable goal for future tumor therapy. Using recombinant adeno-associated virus type 2 (AAV-2) vectors, two types of therapeutic genes were expressed in cervical carcinoma cells with the aim of suppressing the E6/E7 oncogenes: (a) antisense E6/E7 and ribozyme genes and (b) the monocyte chemoattractant protein-1 (MCP-1) gene encoding MCP-1. Previous studies have shown that the MCP-1 protein is able to indirectly repress E6/E7 gene expression and is consistently absent in tumorigenic HPV-positive cervical carcinoma cell lines. Here, the effect of these therapeutic genes on tumor formation is analyzed in nude mice after ex vivo gene transfer into a HPV16- or HPV18-positive cervical carcinoma cell line (HeLa or SiHa, respectively). Whereas AAV-2 vector-mediated transfer of antisense or even ribozyme genes did not significantly influence tumor formation from implanted SiHa cells, the transfer and expression of human MCP-1 strongly inhibited the development of tumors derived from either HeLa or SiHa cells. Similar results were also obtained after in vivo delivery of these genes into SiHa-derived tumors. This suggests that transfer of therapeutic genes mediating a systemic effect via recombinant AAV-2 vectors offers a promising approach for the development of gene therapies directed against papillomavirus-induced human cancers.

Caffeine-derived N-nitroso compounds. V. Carcinogenicity of mononitrosocaffeidine and dinitrosocaffeidine in bd-ix rats.

Mononitrosocaffeidine (MNC) and dinitrosocaffeidine (DNC) are new N-nitroso compounds obtained from in vitro nitrosation of caffeidine, a hydrolysis product of caffeine present in a typically made and widely consumed tea from Kashmir (India), a high incidence area of esophageal and stomach cancer. The chemical synthesis, in vitro metabolic studies and mutagenicity of the compounds has been previously reported. DNC, a nitrosamide is highly mutagenic both with and without metabolic activation whereas MNC, like several other aromatic asymmetric nitrosamines, does not exhibit genotoxic or mutagenic properties. We now report the results of the first carcinogenicity experiments on chronic oral administration of these compounds in BD-IX rats. The acute LD50 of MNC and DNC were about 1300 and 230 mg/kg b.w., respectively. Lung oedema and gastrointestinal haemorrhages were the first symptoms of intoxication observed after 2 days for both the compounds. All three dose groups of MNC treated rats showed localization of tumours in nasal cavity (93.9-100% of all malignant tumours). The tumours were histologically diagnosed as neuroepitheliomas of the olfactory epithelium (neuroblastoma of the bulbus olfactorii) and squamous cell carcinoma of the nasal cavity in the ratio of 3:1. No tumours of the nasal cavity were observed in the untreated controls. DNC, in contrast, induced squamous cell carcinoma of forestomach in 100% animals at low and high doses, of which nearly half the tumours metastasized predominantly into the peritoneum. No forestomach tumours were seen in the untreated controls. The data presented here clearly show the potential for induction of malignant tumours and distinct organ-specificity by MNC and DNC in rats, and support the postulate that a chronic exposure to these compounds may provide a carcinogenic risk for high incidence of gastrointestinal cancers in Kashmir.

Using gene carrier probability to select high risk families for identifying germline mutations in breast cancer susceptibility genes.

Germline mutations in highly penetrant autosomal dominant genes explain about 5% of all breast cancer, and heritable mutations in the BRCA1 breast and ovarian cancer susceptibility gene account for 2-3% of breast cancer in the general population. Nevertheless, the presence of such mutations is highly predictive of disease development. Since screening for mutations is still technically laborious, we investigated whether the prior probability of being a carrier of a dominant breast cancer susceptibility gene in the youngest affected family member could be used to identify families in which the probability of finding a mutation is sufficiently high. Sixty German families with three or more cases of breast/ovarian cancer with at least two cases diagnosed under the age of 60 were screened for mutations by SSCP/CSGE and subsequent direct sequencing. Thirteen germline truncating/splicing mutations in BRCA1 were found in 33% (6/18) of the breast-ovarian cancer families and in 17% (7/42) of breast cancer only families. All the families showing mutations in BRCA1 had carrier probabilities of 0.65 or higher. In families with prior carrier probabilities above 0.6, the proportion detected was 0.46 in breast-ovarian cancer families and 0.26 in breast cancer only families. The average age at diagnosis of breast or ovarian cancer in families with BRCA1 mutations was 41.9 years and significantly lower than in families without mutations (p < 0.05). Mutation carriers and obligate carriers were also found to have cancers at other sites. The probability of being a susceptibility gene carrier, taking into account the complete pedigree information, allows uniform characterisation of all types of families for identifying those in which mutation analysis for BRCA1/2 is warranted. However, prior probabilities calculated using this method can be reduced when the correlation between genotype and phenotype is imperfect. A larger series of families needs to be investigated in this fashion to provide better estimates of the detection rate for different ranges of carrier probabilities.

The expression of the imprinted genes H19 and IGF-2 in choriocarcinoma cell lines. Is H19 a tumor suppressor gene?

H19 is a paternally imprinted gene with unknown function. It is located in close proximity to the maternally imprinted IGF-2 gene on chromosome 11p15.5. In this study no consistent relationship between the expression of these two genes in clones derived from JEG-3 and JAr cell lines could be detected. Nor could a consistent relationship be detected between the expression levels of these two genes and between certain characteristic tumorigenic properties of these clones. We included in this study clones, expressing low H19 levels, which after transfection with an H19 expression construct highly expressed the H19 gene. In tumors, formed by the injection of cells of JAr or JEG-3 clones into nude mice, the H19 expression was high and irrelevant to the expression level in the cells before the injection. The same phenomenon was found for IGF-2 expression during tumorigenesis caused by cells of different JEG-3 clones and in some but not all JAr derived clones. Both H19 and IGF-2 are biallelicly expressed in all the JAr and JEG-3 clones. In summary, our observations point to the conclusion that H19 is not a tumor suppressor gene. However, its high expression in all the tumors formed after injection of cells of the JAr and JEG-3 clones, leaves its role, if any, in choriocarcinogenesis an open question.

The product of the imprinted H19 gene is an oncofetal RNA.

AIMS/BACKGROUND: The H19 gene is an imprinted, maternally expressed gene in humans. It is tightly linked and coregulated with the imprinted, paternally expressed gene of insulin-like growth factor 2. The H19 gene product is not translated into protein and functions as an RNA molecule. Although its role has been investigated for more than a decade, its biological function is still not understood fully. H19 is abundantly expressed in many tissues from early stages of embryogenesis through fetal life, and is down regulated postnatally. It is also expressed in certain childhood and adult tumours. This study was designed to screen the expression of H19 in human cancer and its relation to the expression of H19 in the fetus. METHODS: Using in situ hybridisation with a [35S] labelled probe, H19 mRNA was detected in paraffin wax sections of fetal tissues from the first and second trimesters of pregnancy and of a large array of human adult and childhood tumours arising from these tissues. RESULTS: The H19 gene is expressed in tumours arising from tissues which express this gene in fetal life. Its expression in the fetus and in cancer is closely linked with tissue differentiation. CONCLUSIONS: Based on these and previous data, H19 is neither a tumour suppressor gene nor an oncogene. Its product is an oncofetal RNA. The potential use of this RNA as a tumour marker should be evaluated.

Epitope mapping and functional characterization of monoclonal antibodies specific for herpes simplex virus type I DNA polymerase.

Three monoclonal antibodies (MAbs 1051a, 1051b and 1051c) were raised against a surface region (residues 597-686) of the herpes simplex virus type 1 (HSV-1) DNA polymerase (HSV pol), and their epitopes were mapped. The MAbs reacted serotype specifically with the native and denatured HSV pol, as shown by Western blot analysis, immunoprecipitation and immunofluorescence microscopy, indicating their usefulness for biochemical studies and clinical diagnosis of HSV-1 infections, MAb 1051c, displaying the least cross-reactivity with cellular proteins in the Western blot analysis, was successfully utilized not only for coimmunoprecipitation, but also for the analysis and three-dimensional modeling of the cellular sites of HSV pol interaction by confocal laser immunofluorescence microscopy.

Latent chromosomal instability in cancer patients.

There is increasing evidence that, similar to what is found with other genetic disorders, genomic instability is one of the most general features of cancer. Different forms of manifestation including latent instability have been suggested. To recognize latent chromosomal instability we treated lymphocyte cultures of cancer patients and healthy persons with caffeine, two different doses of bleomycin, and a combination of bleomycin and caffeine. The preliminary results demonstrate that, although the rate of spontaneous chromosomal aberrations is similar in both investigated groups, the lymphocytes of cancer patients display an increased susceptibility to treatment with bleomycin and caffeine. In distinguishing between healthy individuals and those with malignancy, treatment with 30 micrograms/ml bleomycin appears to be most important. Values of chromosomal change above one break per cell, more than 45% cells with chromosomal alterations, and more that two cells with chromosomal rearrangements are suggestive of malignancy. These findings imply that treatment of lymphocyte cultures with bleomycin and caffeine could be a useful assay for monitoring chromosomal instability, and thus detecting a predisposition to malignant disease. In this respect further investigations on a greater amount of material should be performed.

Morphologic characteristics of the interaction between normal cytotrophoblasts and their malignant counterpart in the development of trophoblastic neoplasia.

OBJECTIVES: To define the biology of the tumor-host cell interaction with regard to cellular kinetics and morphologic changes during cell-cell interaction in an in vitro model of trophoblastic neoplasia. METHODS: Using a coculture in vitro system of cytotrophoblasts and choriocarcinoma cells, we investigated the cellular kinetics and the morphologic changes in these interacting cells. A fully automatic time-lapse image system was used to record phase contrast images of the cocultured cells in a tissue culture chamber. To examine cytoskeletal structure, immunofluorescent-labeled antibodies against intermediate filaments were used. Slides were examined with a confocal laser scanning microscope and subjected to computed analysis. RESULTS: The choriocarcinoma cells attract normal cytotrophoblasts using what resembles pseudopodia to engulf the latter cells and thus form slow-growing colonies. In this process, new hybrid cells are formed, which can be differentiated from their original contributors by morphologic characteristics. CONCLUSION: This phenomenon supports our previous biochemical and molecular data on the role of cell-cell interaction in the complex process of cytotrophoblast transformation and the development of gestational trophoblastic neoplasia.

Developmentally imprinted genes as markers for bladder tumor progression.

PURPOSE: Developmentally imprinted genes, such as H19 and insulin-like growth factor-II (IGF-II), play an important role during human embryogenesis and also have been implicated in the pathogenesis of embryonal tumors of childhood. Since H19 is expressed in human fetal bladder, we evaluated 35 bladder carcinomas for H19 expression by in situ hybridization analysis and correlated expression with tumor grade. As a prelude to gene transfer studies to determine if H19 is a bladder tumor oncogene, we also evaluated bladder cell lines for expression of H19, IGF-II, IGF-I and the type I IGF receptor. MATERIALS AND METHODS: H19 expression was evaluated by in situ hybridization analysis in bladder tumor specimens. Northern analysis was used to evaluate the expression of H19, IGF-II, IGF-I and the type I IGF receptor in bladder cell lines. RESULTS: H19 was expressed preferentially in advanced stage tumors: 2 of 12 grade I tumors were H19 positive, whereas 9 of 11 grade II and 7 of 10 grade III tumors expressed H19 (p = 0.004). Additionally, 6 of 6 carcinoma in situ tumors were H19 positive, whereas normal bladder mucosa cells were H19 negative. We found that 3 of 11 cell lines (HT-1376, HT-1197 and 5637) express high levels of H19 mRNA, and each of these cell lines and J82 also express IGF-II. All cell lines examined expressed the type I IGF receptor, whereas there was no detectable IGF-I mRNA. CONCLUSIONS: These data demonstrate that H19 is an oncodevelopmental marker of bladder tumor progression and raise the possibility that H19 may have oncogenic properties in bladder cancer.

Allelic imbalance on chromosome 13q: evidence for the involvement of BRCA2 and RB1 in sporadic breast cancer.

Recently, the breast cancer susceptibility gene BRCA2 has been identified in chromosome 13q, a region that also contains the retinoblastoma gene RB1. To elucidate a possible role of BRCA2 and RB1 in sporadic breast tumorigenesis, allelic imbalance (AI) at 13q loci was examined in 78 primary sporadic breast tumors. AI was found in 52-63% of tumors. Nine tumors showed AI only in the BRCA2 region but not at RB1. Six tumors showed AI at RB1 but not in the BRCA2 region. AI in the BRCA2 region correlated significantly with aneuploidy (P = 0.032) and AI at RB1 with small tumor size (P = 0.025). Our data suggest that BRCA2 and RB1 may be both distinct target loci for AI on chromosome 13 in sporadic breast cancer.

The effect of the JE (MCP-1) gene, which encodes monocyte chemoattractant protein-1, on the growth of HeLa cells and derived somatic-cell hybrids in nude mice.

To investigate the effect of tumor-associated macrophages on the in vivo growth properties of cervical carcinoma cells, tumorigenic human papilloma virus (HPV) 18-positive HeLa cells were transfected with an expression vector harboring the cDNA for the macrophage chemoattractant protein-1 JE (MCP-1). Although the endogenous gene is present and not structurally rearranged, its expression seems to be negatively affected by a still unknown mechanism. Inoculation of JE (MCP-1)-negative HeLa cells into nude mice led to rapidly growing tumors, where macrophage infiltration into the inner tumor mass was not detectable immunohistochemically. The activity that attracted mononuclear cells under both in vitro and in vivo condition was reconstituted in HeLa cells after transfection with the JE (MCP-1) expression vector. Heterotransplantation of those cells into immunocompromised animals resulted in significant growth retardation that was accompanied by a strong infiltration of macrophages. On the other hand, in vivo selection of nonmalignant hybrids made between wild-type HeLa cells and normal human fibroblasts in nude mice resulted in tumorigenic segregants 4 mo after inoculation into the animals. Monitoring JE (MCP-1) expression directly within those nodules, we found that transcription was either absent or only weakly detectable. Recultivation of JE (MCP-1)-positive tissue grafts under in vitro conditions revealed that the gene was only marginally inducible by tumor necrosis factor-alpha, a cytokine that normally induces a very strong activation of transcription in nontumorigenic cells. These findings suggest that functional JE (MCP-1) expression and in turn activated macrophages may play a pivotal role in controlling the proliferation rate of HPV-positive cells in vivo.

Trisomy 8 and urogenital malformation in the ACI-rat.

ACI-rats are considered as a model for studying urogenital abnormalities. In order to recognise cytogenetic changes related to these abnormalities 50 male ACI/Seg rats were examined by means of gross macroscopic, histological, and karyotypical investigations. In six of the examined animals (12%) unilateral agenesis of the kidney and ipsilateral hypoplasia of the testes and seminal vesicles were observed. Isochromosome 8 and trisomy 8 (i8, +8) were observed in 26.5% of karyotypes from the animals with kidney agenesis. Chromosome heteromorphisms such as 1p+, 3p+, 11p+, 12p+ were found in animals with and without apparent pathology. Because of the similarity between the phenotypical changes found in ACI-rats and in patients with familial renal agenesis (Potter's syndrome) and hereditary renal agenesis and aplasia (HRA), rat and human chromosomes associated with manifested renal malformations were examined by comparative cytogenetics and gene mapping.

Evaluation of nuclear morphology in adriamycin sensitive and resistant cells.

The adriamycin sensitive and resistant human breast cancer cells (MCF-7) were investigated by computerized image analysis for establishment of characteristic cell nuclei-morphological differences. Using specially developed algorithms and a large set of subvisual parameters, morphological features characteristic for the resistant cell line were described. The most useful parameters for distinguishing the resistant from sensitive cells are related to chromatin structure and include integral optical density per micron 2, the average area of chromatin region, and the integral density of the chromatin region per micron 2. Besides the size of the nuclei, the two features with the most discriminatory power belonged to those characterizing patterns of chromatin condensation. The results of quantitative analysis suggest that drug resistance relates to specific chromatin patterns. Compared to sensitive cell line, the resistant MCF-7 cells show distinguished coarse chromatin pattern with enlarged condensed chromatin regions.

Genetic alterations in sporadic renal-cell carcinoma: molecular analyses of tumor suppressor gene harboring chromosomal regions 3p, 5q, and 17p.

On a genetic level, renal-cell carcinoma has been characterized by an abnormality on the short arm of chromosome 3 (3p), which suggests the inactivation of a tumor suppressor gene. One tumor suppressor gene at 3p, the von Hippel-Lindau disease gene, is implicated in tumor development of a whole spectrum of hereditary neoplasms, including renal-cell carcinoma. It is not clear whether the same tumor suppressor gene accounts for all, i.e., hereditary and sporadic, renal-cell carcinomas. Analysis of 28 patients with sporadic renal-cell carcinomas for loss of heterozygosity was performed at chromosomal regions that contain known tumor suppressor genes so as to assess their potential involvement during renal tumorigenesis. We focused on chromosome 3p because it contains the von Hippel-Lindau (VHL) disease gene, on 5q because it harbors tumor suppressor genes involved in colorectal carcinoma, and on 17p because it includes a tumor suppressor gene involved in breast, colon, and lung carcinoma. Loss of alleles at 3p affected 96% of the evaluable patients, with frequencies being highest in the VHL region in 3p25-26 and at loci in 3p21. These data confirm the importance of a 3p defect early during tumorigenesis; however, the question as to the existence of a second renal-cell carcinoma gene remains unresolved. Changes at 5q were 53% and those at 17p were 35%, suggesting that these loci may not contribute to the initiation of the disease but rather may represent accumulating genetic defects associated with progression and malignancy.

Subclinical human immunodeficiency virus-related changes in oral mucosa shown by image analysis of tongue smears.

There is evidence that certain lesions of the oral mucosa, such as hairy leukoplakia (HL), in patients seropositive for human immunodeficiency virus correlate with the subsequent development of acquired immune deficiency syndrome. The authors suggest that HL is a final manifestation of alterations that gradually develop after HIV infection. To recognize inapparent early subclinical changes in oral mucosa, the authors applied methods of digital image analysis to investigate tongue smears from healthy control subjects and immunosuppressed patients after chemotherapy and HIV infection. Their studies concentrated on nuclear morphologic features and chromatin structure. The results obtained with a large set of subvisual parameters indicated significant differences in nuclear and chromatin features between the smear patterns of investigated groups. One important implication of these studies is that computerized image analysis of simply prepared tongue smears enables one to recognize subvisual HIV-related changes before clinical evidence of HL appears.

Chromatin organization and breast cancer prognosis. Two-dimensional and three-dimensional image analysis.

BACKGROUND: The assessment of breast cancer prognosis still relies on clinical staging and histologic grading. Among the different prognostic parameters, attention has been focused on features of chromatin structure. Although data from two-dimensional (2-D) analysis of chromatin structure show significant correlation with the survival, their biologic interpretation remains difficult. In this respect, insights into the spatial organization of chromatin appear important. METHODS: The authors performed computerized three-dimensional (3-D) reconstructions of Feulgen-stained tumor cell nuclei, correlating the results with those of 2-D image analysis. Forty-nine ductal-invasive carcinomas with 10-18 years follow-up were investigated. RESULTS: The 2-D analysis demonstrated that along with nuclear size and shape, chromatin features such as marginal concentration of condensed chromatin are of prognostic importance. As 3-D analysis revealed, chromatin changes concern ordered organization and were associated with the increase of spatial entropy. CONCLUSIONS: The 3-D description of chromatin structure by means of spatial models provides evidence that tumor aggressiveness correlates with events of clustering and randomization. These indicate disturbances of ordered chromatin organization.

Steroidal interactions in the ageing endocrine system: absence of suppression and pathology in reproductive systems of old males from a mixed-sex socially stressful rat colony.

A paradigm using chronic social stress and multiple measures of the reproductive system were used to assess changes with ageing in the dynamics of endogenous steroid interactions. The 22- to 24-month-old male rats lived for 8 weeks in one of four types of colony, in groups of the same sex or groups of mixed sex including familiar or unfamiliar old males. Measures of endocrinology (circulating steroid levels), behaviour (exploration and sociosexual responses), physiology (body and organ weights and epididymal sperm count) and histology (adrenal and ventral prostate glands) served as markers of activation of the hypothalamic-pituitary-adrenal (HPA) or hypothalamic-pituitary-testicular (HPT) axes. Old males living under stable conditions as familiar same-sex colonies served as the comparison group. Results indicated clear chronic activation of the HPA axis in the unfamiliar all-male colonies and of the HPT axis in the familiar males from mixed-sex colonies, whereas both steroidal axes were stimulated in colonies of unfamiliar males and females. Findings from aged males under chronic stress suggested that reproductive dysfunction may be limited to situations in which activation of the HPA axis occurs without concurrent stimulation of the HPT axis. Data on steroidal interactions from mixed-sex groups suggested that (1) chronic excitation of the HPA failed to suppress function in the reproductive system of the old males, (2) their stress responses were little affected by chronic HPT activation and (3) there was no evidence for stress-induced pathology, even in the vulnerable prostate gland.(ABSTRACT TRUNCATED AT 250 WORDS)

Prevention of tumor metastasis formation by anti-variant CD44.

A splice variant of CD44 (CD44v) originally discovered on metastases of a rat pancreatic adenocarcinoma (BSp73ASML) has been shown by transfection to confer metastatic behavior to nonmetastatic tumor cells (Günthert U., M. Hofmann, W. Rudy, S. Reber, M. Zöller, I. Haussmann, S. Matzku, A. Wenzel, H. Ponta, and P. Herrlich. 1991. Cell. 65:13). A monoclonal antibody (mAb), 1.1ASML, to the metastasis-specific domain of the CD44v molecule retards growth of lymph node and lung metastases of the metastatic tumor line BSp73ASML, and can efficiently prevent formation of metastases by the transfected line. The antibody is only effective when given before lymph node colonization. Anti-CD44v does not downregulate the expression of CD44v, and prevention of metastatic growth by anti-CD44v is not due to activation of any kind of immune defense. We suggest that the mAb interferes with proliferation of metastasizing tumor cells in the draining lymph node, most probably by blocking a ligand interaction. The interference with metastatic spread will greatly facilitate the exploration of the function of CD44v and, in particular, may also open new strategies for the therapy of human metastases.

Growth of Hodgkin cell lines in severely combined immunodeficient mice.

No animal model exists for the in vivo growth of Hodgkin's-lymphoma-derived cells. Neither unmanipulated Hodgkin's-disease(HD)-derived cell lines nor primary biopsy tissue could be grown in nude mice. Since the severe combined immunodeficient (SCID) mouse has been reported to be a better recipient for transplanted human lymphatic tissue than the nude mouse, we tested whether SCID mice provide suitable conditions for the in vivo growth of HD cell lines. Tumorigenicity of HD cells was tested in untreated and pre-treated SCID mice and in another combined immunodeficient mouse strain, beige/nude/X-linked immunodeficient (BNX) mouse. SCID mice supported in vivo growth of the 6 HD cell lines tested (L428, L540, L591, DEV, HD-LM2, KM-H2). Only one of the 6 lines (DEV) was tumorigenic in BNX mice. No HD cell line proliferated in T-cell-deficient nude mice. Thus, in vivo growth of HD cell lines appeared to be related to the degree of host immunodeficiency. Additional growth supportive treatments such as fibrosarcoma co-transplantation, intraperitoneal mineral oil injection or immunosuppressive pre-treatment (anti-asialo-GMI-antibody injection) permitted growth of 3 additional HD cell lines in BNX mice. The immunophenotype and karyotype of explanted graft cells were identical to the original cell lines. Our experiments describe an effective and reproducible xenograft model for growth of Hodgkin's-disease-derived cell lines. This may be of value for elucidating the growth characteristics of Hodgkin's-lymphoma-derived cells as well as for testing new therapeutic regimens.