Induction Of Malignant Transformation Research Articles

Untargeted metabolomics reveals alternations in metabolism of bovine mammary epithelial cells upon IFN-γ treatment

BackgroundIFN-γ is a pleiotropic cytokine that has been shown to affect multiple cellular functions of bovine mammary epithelial cells (BMECs) including impaired milk fat synthesis and induction of malignant transformation via depletion of arginine, one of host conditionally essential amino acids. But the molecular mechanisms of these IFN-γ induced phenotypes are still unknown.MethodsBMECs were treated with IFN-γ for 6 h, 12 h, and 24 h. The metabolomic profiling in BMECs upon IFN-γ induction were assessed using untargeted ultra-performance liquid chromatography-mass spectrometry (UPLC-MS) metabolomic analysis. Key differentially expressed metabolites (DEMs) were quantified by targeted metabolomics.ResultsIFN-γ induction resulted in significant differences in the contents of metabolites. Untargeted analysis identified 221 significantly DEMs, most of which are lipids and lipid-like molecules, organic acids and derivatives, organ heterocyclic compounds and benzenoids. According to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis, DEMs were enriched in fructose and mannose metabolism, phosphotransferase system (PTS), β-alanine metabolism, arginine and proline metabolism, methane metabolism, phenylalanine metabolism, and glycolysis/gluconeogenesis. Quantification of selected key DEMs by targeted metabolomics showed significantly decreased levels of D-(-)-mannitol, argininosuccinate, and phenylacetylglycine (PAG), while increased levels in S-hydroxymethylglutathione (S-HMG) and 2,3-bisphospho-D-glyceric acid (2,3-BPG).ConclusionsThese results provide insights into the metabolic alterations in BMECs upon IFN-γ induction and indicate potential theoretical basis for clarifying IFN-γ-induced diseases in mammary gland.

MRI detection of the malignant transformation of stem cells through reporter gene expression driven by a tumor-specific promoter

BackgroundExisting evidence has shown that mesenchymal stem cells (MSCs) can undergo malignant transformation, which is a serious limitation of MSC-based therapies. Therefore, it is necessary to monitor malignant transformation of MSCs via a noninvasive imaging method. Although reporter gene-based magnetic resonance imaging (MRI) has been successfully applied to longitudinally monitor MSCs, this technique cannot distinguish the cells before and after malignant transformation. Herein, we investigated the feasibility of using a tumor-specific promoter to drive reporter gene expression for MRI detection of the malignant transformation of MSCs.MethodsThe reporter gene ferritin heavy chain (FTH1) was modified by adding a promoter from the tumor-specific gene progression elevated gene-3 (PEG3) and transduced into MSCs to obtain MSCs-PEG3-FTH1. Cells were induced to undergo malignant transformation via indirect coculture with C6 glioma cells, and these transformed cells were named MTMSCs-PEG3-FTH1. Western blot analysis of FTH1 expression, Prussian blue staining and transmission electron microscopy (TEM) to detect intracellular iron, and MRI to detect signal changes were performed before and after malignant transformation. Then, the cells before and after malignant transformation were inoculated subcutaneously into nude mice, and MRI was performed to observe the signal changes in the xenografts.ResultsAfter induction of malignant transformation, MTMSCs demonstrated tumor-like features in morphology, proliferation, migration, and invasion. FTH1 expression was significantly increased in MTMSCs-PEG3-FTH1 compared with MSCs-PEG3-FTH1. Prussian blue staining and TEM showed a large amount of iron particles in MTMSCs-PEG3-FTH1 but a minimal amount in MSCs-PEG3-FTH1. MRI demonstrated that the T2 value was significantly decreased in MTMSCs-PEG3-FTH1 compared with MSCs-PEG3-FTH1. In vivo, mass formation was observed in the MTMSCs-PEG3-FTH1 group but not the MSCs-PEG3-FTH1 group. T2-weighted MRI showed a significant signal decrease, which was correlated with iron accumulation in the tissue mass.ConclusionsWe developed a novel MRI model based on FTH1 reporter gene expression driven by the tumor-specific PEG3 promoter. This approach could be applied to sensitively detect the occurrence of MSC malignant transformation.

Signal Activation of Hepatitis B Virus-Related Hepatocarcinogenesis by Up-regulation of SUV39h1.

Hepatitis B virus (HBV) X (HBx) protein is associated with hepatocellular carcinogenesis via the induction of malignant transformation and mitochondrial dysfunction. However, the association between HBx and histone methyltransferase in carcinogenesis has not been fully clarified. In the current study, we analyzed the association between HBx and the histone methyltransferase suppressor of variegation 3-9 homolog 1 (SUV39h1) using HBV replication models. We constructed several HBx and SUV39h1 expression plasmids and analyzed the association between HBx and SUV39h1 with respect to HBV replication and hepatocarcinogenesis. SUV39h1 up-regulation was observed in HBV-infected humanized mouse livers and clinical HBV-related hepatocellular carcinoma tissues, indicating that SUV39h1 expression might be regulated by HBV infection. Through in vitro analysis, we determined that the coactivator domain of HBx interacts with the PSET (PostSET) and SET (Su(var)3-9, Enhancer-of-zeste, Trithorax) domains of SUV39h1. The expression levels of 4 genes, activating transcription factor 6, α-fetoprotein, growth arrest and DNA damage-inducible 45a, and dual-specificity phosphatase 1, known to induce carcinogenesis via HBx expression, were up-regulated by HBx and further up-regulated in the presence of both HBx and SUV39h1. Furthermore, histone methyltransferase activity, the main function of SUV39h1, was enhanced in the presence of HBx. We demonstrated that SUV39h1 and HBx enhance each other's activity, leading to HBx-mediated hepatocarcinogenesis. We propose that regulation of this interaction could help suppress development of hepatocellular carcinoma.

MicroRNAs in virus-induced tumorigenesis and IFN system

Numerous microRNAs (miRNAs), small non-coding RNAs encoded in the human genome, have been shown to be involved in cancer pathogenesis and progression. There is evidence that some of these miRNAs possess proapoptotic or proliferation promoting roles in the cell by negatively regulating target mRNAs. Oncogenic viruses are able to produce persistent infection, favoring tumor development by deregulating cell proliferation and inhibiting apoptosis.It has been recently suggested that cellular miRNAs may participate in host–virus interactions, influencing viral replication. Many mammalian viruses counteract this cellular antiviral defense by using viral proteins but also by encoding viral miRNAs involved in virus-induced tumorigenesis.Interferons (IFNs) modulate a number of non-coding RNA genes, especially miRNAs, that may be used by mammalian organisms as a mechanism of IFN system to combat viral infection and related diseases. In particular, IFNs might induce specific cellular miRNAs that target viral transcripts thereby using this strategy as part of their effectiveness against invading viruses. Therefore IFNs, interferon stimulated genes and miRNAs could act synergistically as innate response to virus infection to induce a potent non-permissive cellular environment for virus replication and virus-induced cancer.The relevance of this reviewed research topic is clearly related to the observation that although virus infections are responsible of specific tumors, other unidentified genetic alterations are likely involved in the induction of malignant transformation. The identification of such genetic alterations, i.e. miRNA expression in transformed cells, would be of considerable importance for the analysis of the pathogenesis and for the treatment of cancer induced by specific viruses as well as for the advancement of the current knowledge on the molecular mechanisms underlying virus–host interaction. In this respect, we will review also the important, still little explored, roles of miRNAs acting both as IFN-stimulated anti-viral molecules and as critical regulators of IFNs and IFN-stimulated genes.

Long isoform of ErbB3 binding protein, p48, mediates protein kinase B/Akt-dependent HDM2 stabilization and nuclear localization

p48 is a long isoform of the ErbB3 binding protein that has oncogenic functions including promotion of carcinogenesis and induction of malignant transformation through negative regulation of tumor suppressor p53. Here, we show that high level of p48 protein expression leads to enhance HDM2 phosphorylation by Akt and inhibits the self-ubiquitination of HDM2 by up-regulation of Akt activity, thereby promoting its protein stability. Moreover, p48 expression leads to accumulated nuclear localization of HDM2, whereas p48 depletion disturbs its nuclear localization. Hence, higher expression of p48 in cancer cells reduces p53 levels through modulation of HDM2 nuclear localization and protein stability via regulation of its Akt-mediated phosphorylation.

Involvement of Autophagy in Oncogenic K-Ras-induced Malignant Cell Transformation

Autophagy has recently been implicated in both the prevention and progression of cancer. However, the molecular basis for the relationship between autophagy induction and the initial acquisition of malignancy is currently unknown. Here, we provide the first evidence that autophagy is essential for oncogenic K-Ras (K-RasV12)-induced malignant cell transformation. Retroviral expression of K-RasV12 induced autophagic vacuole formation and malignant transformation in human breast epithelial cells. Interestingly, pharmacological inhibition of autophagy completely blocked K-RasV12-induced, anchorage-independent cell growth on soft agar. Both mRNA and protein levels of ATG5 and ATG7 (autophagy-specific genes 5 and 7, respectively) were increased in cells overexpressing K-RasV12. Targeted suppression of ATG5 or ATG7 expression by short hairpin (sh) RNA inhibited cell growth on soft agar and tumor formation in nude mice. Moreover, inhibition of reactive oxygen species (ROS) with antioxidants clearly attenuated K-RasV12-induced ATG5 and ATG7 induction, autophagy, and malignant cell transformation. MAPK pathway components were activated in cells overexpressing K-RasV12, and inhibition of JNK blunted induction of ATG5 and ATG7 and subsequent autophagy. In addition, pretreatment with antioxidants completely inhibited K-RasV12-induced JNK activation. Our results provide novel evidence that autophagy is critically involved in malignant transformation by oncogenic K-Ras and show that reactive oxygen species-mediated JNK activation plays a causal role in autophagy induction through up-regulation of ATG5 and ATG7.

Contact by melanoma cells causes malignant transformation of human epithelial-like stem cells via alpha V integrin activation of transforming growth factor β1 signaling

The embryonic microenvironment is known to suppress the tumorigenic phenotype of aggressive cancer cells; however, the effects of tumorigenic microenvironments on stem cells have not been sufficiently explored due to the lack of suitable model systems. In order to study the tumorigenic microenviornment, we developed a novel in vitro model system for induction of malignant transformation of human epithelial-like stem cells (hEpSCs), involving co-cultivation and close contact of hEpSCs with the A375 melanoma cell line, together with mutagen treatment of hEpSCs with dimethylbenzanthracene (DMBA). Both factors (close contact and mutagen treatment) were required to transform hEpSCs in vitro and cause phenotypic changes characteristic of epithelial to mesenchymal transition (EMT), including colony formation, decreased E-cadherin and increased N-cadherin and vimentin expression. Direct contact between tumor cells and hEpSCs treated with DMBA increased integrin alpha V (ITGAV gene) expression and caused local activation of the transforming growth factor (TGF)-β1/Smad signaling pathways in hEpSCs. The novel model system described here is being used to elucidate the microenvironmental factors and biological mechanisms involved in the induction of neoplastic progression in hEpSCs in vitro by A375 melanoma cells. A better understanding of the molecular mechanisms by which melanoma cells exert these effects on hEpSCs may open up new avenues for therapeutic and preventive cancer interventions.

Relationship among human papillomavirus infection, p16INK4a, p53 and NF-κB activation in penile cancer from northern Thailand

Human papillomavirus (HPV) E6 and E7 oncoproteins are essential factors for HPV oncogenesis. These E6 and E7 gene products play a central role in the induction of malignant transformation by interacting with several cellular regulatory proteins such as p16(INK4a), p53 and nuclear factor κB (NF-κB). In the present study, conducted in northern Thailand, HPV-DNA was detected in penile cancer cases using an in situ hybridization procedure and p16(INK4a), p53 and NF-κB were detected by immunohistochemistry. Using the cell cycle regulatory proteins p16(INK4a) (61.5%) and p53 (71.8%), it was found that of the 51 cases, 39 (76.5%) were HPV-DNA-positive in penile cancer. On the other hand, 25% p16(INK4a) and 75% p53, respectively, were found in HPV-negative cases. Prevalence of HPV infection (76.5%) was shown in penile cancer cases in northern Thailand. No difference was found between HPV-positive and HPV-negative cases with respect to the presence of the cell cycle regulatory protein p53. On the other hand, p16(INK4a) was found to be different between HPV-positive and HPV-negative cases. Inactivation of tumor suppressor genes, such as p16(INK4a) and p53, to genetic instability, cell immortalization, accumulation of mutations and cancer formation, with or without HPV and irrespective of HPV infection, is therefore suggested. Of the 39 HPV-positive cases, 35 (89.7%) were NF-κB-positive in the nucleus, 29 (74.4%) in the cytoplasm and 37 (94.9%) in the nucleus and/or cytoplasm. NF-κB was detected in 4 (33.3%) of the 12 HPV-negative cases. Therefore, we propose that penile cancer cases with HPV infection are more likely to activate NF-κB than those without HPV infection.

High Incidence of Hepatitis B or C Virus Infection in Lymphoma.

Abstract 3934Poster Board III-870 IntroductionIn lymphoma, several reports from different countries have shown that patients with lymphoma have a higher prevalence of HCV or HBV infection. The infection rates were very different even in virus endemic areas as Asia-Pacific countries including Japan. However, an association between virus infection and lymphoma should be evaluated and discussed with the background of virus prevalence in the community. This study aimed to investigate the prevalence of HBV or HCV infection status in lymphoma patients in Tohoku distinct in Japan. Patients and MethodsThe study, designed by the Tohoku Hematology Forum Group, was conducted in 9 clinical centers in Tohoku Distinct in Japan. This study included 1331 newly diagnosed consecutive series of lymphoma patients admitted in Tohoku Hematology Forum Group Hospital (Japan) and 848 non-lymphoma control patients enrolled from January 2005 to October 2008. We evaluated the prevalence of HBV (as indicated by hepatitis B surface antigen) or HCV infection in both group patients and compared the clinic-pathological characteristics of HBV- or HCV-positive and its negative lymphoma cases. ResultsThe carrier rate of HBsAg was higher in lymphoma patients (57 of 1331, 4.3%) than control group (1.7%, p <0.001) The higher prevalence was observed in both sexes and especially diffuse large B cell lymphoma. Compared with HBV negative lymphoma group, the positive group displayed more liver or spleen involvement (p=0.002). The rate of HCV infection was higher than those in control group In HCV-infected case, more liver involvement was indicated (p<0.004). No difference in HBV or HCV prevalence was found between B-cell and T/NK-cell lymphoma. ConclusionsThe present study, so far the largest trial of incidence of HBV or HCV infection in lymphoma patients, demonstrated that patients with lymphoma in Japan had higher prevalence of HBV and HCV infection. These appears to be a possible role for HBV or HCV in the induction of malignant transformation, resulting in the development of lymphoma. However, the precise role of HBV or HCV in the pathogenesis of lymphoma remains to be elucidated and warrants further research. Disclosures:No relevant conflicts of interest to declare.

Localization of tissue inhibitor of metalloproteinases 1 (TIMP-1) in human colorectal adenoma and adenocarcinoma.

Tissue inhibitor of matrix metalloproteases 1 (TIMP-1) inhibits the proteolytic activity of matrix metalloproteases and hereby prevents cancer invasion. However, TIMP-1 also possesses other functions such as inhibition of apoptosis, induction of malignant transformation and stimulation of cell-growth. We have previously demonstrated that TIMP-1 is elevated in blood from colorectal cancer patients and that high TIMP-1 levels predict poor prognosis. To clarify the role of TIMP-1 in colorectal tumorigenesis, the expression pattern of TIMP-1 in benign and malignant colorectal tumors was studied. In all of 24 cases of colorectal adenocarcinoma TIMP-1 mRNA was detected by in situ hybridization. In all cases TIMP-1 expression was found in fibroblast-like cells located at the invasive front but was seen only sporadically in normal mucosa. No TIMP-1 mRNA was seen in any of the cases in benign or malignant epithelial cells, in vascular cells or smooth muscle cells. Comparison of sections processed for TIMP-1 in situ hybridization with sections immunohistochemically stained with antibodies against TIMP-1 showed good correlation between TIMP-1 mRNA and immunoreactivity. Combining TIMP-1 in situ hybridization with immunohistochemical staining for alpha-smooth muscle actin or CD68 showed TIMP-1 mRNA in myofibroblasts but not in macrophages. TIMP-1 mRNA was detected in 2 of 7 adenomatous polyps in the adenoma area: in both cases associated with focal stromal inflammation at the epithelial-stromal interface. In conclusion, TIMP-1 expression is a rare event in benign human colon tissue but is highly expressed by myofibroblasts in association with invading colon cancer cells.

Nuclear localization of KLF4 is associated with an aggressive phenotype in early-stage breast cancer.

The Krüppel-like transcription factor KLF4/GKLF induces both malignant transformation and a slow-growth phenotype in vitro. Although KLF4 expression is increased in most cases of breast cancer, it was unknown whether these cases represent a distinct subtype with a different clinical outcome. We examined expression of KLF4 by immunostaining 146 cases of human primary infiltrating ductal carcinoma of the breast. Staining patterns were correlated with clinical outcome and with established prognostic factors. Subcellular localization exhibited case-to-case variation. Tumors with high nuclear staining and low cytoplasmic staining were termed type 1. For patients with early-stage disease (i.e., stage I or IIA), type 1 staining was associated with eventual death because of breast cancer (hazard ratio, 2.8; 95% confidence interval, 1.23-6.58; P = 0.011). The association was stronger in patients with early-stage cancer and small primary tumors (i.e., < or =2.0 cm in diameter; hazard ratio, 4.3; 95% confidence interval, 1.75-10.62; P < 0.001). For patients with early-stage disease, multivariate analysis indicated that type 1 staining was independently associated with outcome (adjusted hazard ratio 2.6; 95% confidence interval, 1.10-6.05; P = 0.029). Type 1 staining was also associated with high histological grade (P = 0.032), increased expression of Ki67 (P = 0.016), and reduced expression of BCL2 (P = 0.032). In vitro, KLF4 was localized within the nucleus of transformed RK3E epithelial cells, consistent with a nuclear function of this transcription factor during induction of malignant transformation. The results suggest that localization of KLF4 in the nucleus of breast cancer cells is a prognostic factor and identify KLF4 as a marker of an aggressive phenotype in early-stage infiltrating ductal carcinoma.

The γ-subunit of the coatomer complex binds Cdc42 to mediate transformation

The Ras-related GTP-binding protein Cdc42 is implicated in a variety of biological activities including the establishment of cell polarity in yeast, the regulation of cell morphology, motility and cell-cycle progression in mammalian cells and the induction of malignant transformation. We identified a Cdc42 mutant (Cdc42F28L) which binds GTP in the absence of a guanine nucleotide exchange factor, but still hydrolyses GTP with a turnover number identical to that for wild-type Cdc42. Expression of this mutant in NIH 3T3 fibroblasts causes cellular transformation, mimicking many of the characteristics of cells transformed by the Dbl oncoprotein, a known guanine nucleotide exchange factor for Cdc42. Here we searched for new Cdc42 targets in an effort to understand how Cdc42 mediates cellular transformation. We identified the gamma-subunit of the coatomer complex (gammaCOP) as a specific binding partner for activated Cdc42. The binding of Cdc42 to gammaCOP is essential for a transforming signal distinct from those elicited by Ras.

Folic acid deficiency and cancer: mechanisms of DNA instability

Folic acid deficiency in humans has been linked with megaloblastic anaemia, neural tube defects in the neonate, and heart disease. Folate has also been implicated in the development of cancer, especially cancer of the colorectum. There appear to be two principal mechanisms through which low folate status may increase the risk of malignancy. Folate deficiency, by reducing intracellular S-adenosylmethionine (SAM), can alter cytosine methylation in DNA, leading to inappropriate activation of proto-oncogenes and induction of malignant transformation. Alternatively, folic acid is crucial for normal DNA synthesis and repair. Folate deficiency may cause an imbalance in DNA precursors, uracil misincorporation into DNA, and chromosome breakage. This chapter briefly describes the epidemiological data supporting the involvement of folic acid in the aetiology of cancer. It also assesses the evidence from cellular, animal and human studies that folic acid can modulate DNA by such mechanisms.

Bcl-2 enhancement of malignant transformation in mouse epidermal JB6 cells.

Increased bcl-2 expression is a common feature of many types of human malignancies, which implies that bcl-2 plays an important role in tumorigenesis. To better understand the molecular mechanisms of bcl-2-induced oncogenesis, we examined the effects of bcl-2 expression on transformation of mouse epidermal JB6 cells induced by the tumor promoter 12-O-tetradecanoylphorbol-13 acetate (TPA). Promotion-sensitive JB6 clone41 cells were transfected with the bcl-2-containing expression vector pD5-neo/bcl-2, and the soft agar growth of bcl-2-transfected cells and control cells were compared. bcl-2 overexpression in JB6 clone41 cells caused a TPA-induced soft-agar growth fivefold greater than the growth of nontransfected or vector-transfected (neo control) cells. bcl-2 expression in the absence of TPA did not lead to colony formation in soft agar. Because the level of the transcription factor activator protein 1 (AP-1) has been shown to be critical for the responsiveness of JB6 cells to TPA-induced transformation, we compared c-jun and c-fos expression as well as the AP-1-binding activity and the AP-1-mediated transactivation of the reporter construct TRE-CAT between bcl-2-expressing cells and control cells. When compared with control cells, bcl-2-transfected cells expressed significantly more c-fos but not c-jun after TPA treatment. Furthermore, the levels of AP-1 and AP-1-induced transactivation of TRE-CAT were greater in bcl-2-transfected cells than in control cells after TPA treatment. These results showed that bcl-2 cooperates with a tumor promoter such as TPA in the induction of malignant transformation in mouse epidermal cells and that bcl-2 enhances soft-agar growth by stimulating signaling pathways that led to increased AP-1 expression.

Radiation-initiated, immortal Syrian hamster embryo fibroblasts release elevated levels of H2O2 and show divergent MnSOD and catalase activities

To characterize molecular events associated with the neoplastic conversion of primary cells by ionizing radiation, we studied the activities and mRNA expression of manganese superoxide dismutase (MnSOD) and catalase (CAT) in Syrian hamster embryo fibroblasts during the early stages of immortalization after treatment with gamma-rays. The irradiated cells showed divergent MnSOD and CAT responses relative to unirradiated controls. At passage 6, MnSOD activity was increased about 50-fold, although the concentration of MnSOD mRNA increased only 1.6-fold. By contrast, CAT activity diminished 2-fold despite an increase of 1.6-fold in the concentration of CAT mRNA. This divergence between the MnSOD and CAT activities was maintained upon culturing and, at passage 12, MnSOD was 35-fold increased and CAT 3.7-fold decreased, relative to unirradiated cells. The amount of H2O2 released into the culture medium by the radiation-initiated cells was 6-fold greater than in control media. Because H2O2 is a causative agent in the induction of malignant transformation in vitro, our results suggest that the elevated production of H2O2 caused by the imbalance between the activities of MnSOD and CAT may participate in the immortalization and subsequent malignant conversion of Syrian hamster embryo fibroblasts by ionizing radiation.

Malignant transformation of human fibroblasts by ionizing radiation.

As one step in developing an assay for quantifying the induction of malignant transformation of human cells by ionizing radiation, we exposed cells from a non-tumorigenic, infinite life span, near-diploid fibroblast strain MSU-1.1 to 4.35 Gy 60Co radiation and assayed them for focus formation. The mean frequency of foci in the irradiated population was 6 x 10(-7) cells assayed. No foci were found in the control cells. Of four focus-derived cell strains studied in detail, two produced malignant tumours within 3-7 weeks. The other two did not produce tumours during the 12-month period of study. The tumours from one strain were classified as sarcomas composed exclusively of spindle-shaped cells. Tumours from the other strain were sarcomas consisting of a mixed population of round and spindle cells. Immunoprecipitation analysis of the status of the p53 gene in the focus-derived strains, using a mutant-specific anti-body (Pab240) and an antibody that recognizes both mutant and wild-type p53 protein (Pab421), showed that the tumorigenic strains were completely devoid of p53 protein. One non-tumorigenic strain expressed wild-type p53 protein, and the other expressed a lower molecular weight form of the protein. Karyotypic analysis showed that the tumour-derived cells from one tumorigenic strain had lost one copy of chromosome 6, 14, 16 and 17. The tumour-derived cells from the second strain had lost one copy of chromosome 7, 13, 14 and 17 and part of chromosome 6, as well as part of the other copy of chromosome 7 and 17. These results suggest that the common loss of one copy of chromosome 14, 17 and part of 6 plays a causal role in the malignant transformation of these cells. Furthermore, the results indicate that it will be possible to develop a system that uses near-diploid human fibroblasts to quantify radiation-induced malignant transformation.

Studies of ionizing radiation as a promoter of neoplastic transformation in vitro.

The induction of malignant transformation was examined in a standard promotion protocol in which BALB/3T3 cells were incubated continuously with tritiated water (3HOH) following acute treatment with various doses of either X-rays or benzo(a)pyrene (BP). In no case was there any evidence that protracted exposure to ionizing radiation from 3HOH enhanced the yield of transformants induced by the primary carcinogen over that predicted if the effects of the two agents were additive.

Induction of malignant transformation in vitro and mammary tumors in rats by two new potent anthracycline antitumor antibiotics, morpholinodaunomycin and cyanomorpholinoadriamycin.

The two new potent anthracycline antitumor antibiotics, morpholinodaunomycin and cyanomorpholinoadriamycin, are non-mutagenic or weakly mutagenic in Salmonella typhimurium or V79 Chinese hamster cells, but highly active inducing DNA repair in in cultured rat hepatocytes. Both agents were found to induce malignant transformation in vitro of C3H M2 mouse fibroblasts and mammary tumors in female Sprague-Dawley rats. The data indicate a) that these new anthracyclines, too, are highly oncogenic and b) in conjunction with previously published results, that the predictive value of in vitro short-term tests for in vivo carcinogenicity is dependent on the employment of a battery of such tests.

Pyrene acts as a cocarcinogen with the carcinogens benzo[a]pyrene, beta-propiolactone and radiation in the induction of malignant transformation in cultured mouse fibroblasts; soybean extract containing the Bowman-Birk inhibitor acts as an anticarcinogen.

Pyrene was found to act as a cocarcinogen in the induction of transformation of cultured Balb/c3T3 cells by three different types of carcinogens: a direct acting chemical carcinogen, beta-propiolactone, a chemical carcinogen requiring metabolic activation, benzo[a]pyrene, and a physical carcinogen (60Co) gamma radiation. Since pyrene enhanced transformation in vitro by approximately the same amount for all the carcinogens tested, these results suggest that the carcinogenic action of pyrene is not related to carcinogen metabolism or uptake in vitro. An extract of soybeans containing the Bowman-Birk protease inhibitor was shown to reduce transformation induced by beta-propiolactone, benzo[a]pyrene and gamma-rays, both with and without the cocarcinogenic effect of pyrene, to background levels; the magnitude of the reduction in transformation by the protease inhibitor preparation was unrelated to the concentration of carcinogen. Neither the mechanism for the cocarcinogenic action of pyrene not the anticarcinogenic effect of the soybean extract is known, but several hypotheses are discussed.

Induction of malignant transformation by various chemicals in Balb/3T3 clone A31-1-1 cells and biological characterization of some transformants

Balb/c A31-1-1 cells were used for the study of transformation induction by chemicals with different mutagenic specificities. We show that survival of these cells and therefore the calculated transformation frequency per cells at risk is dependent upon the cell density at the time of treatment. It is suggested that equal cell densities should be used for measuring survival values and transformation induction. The quantitative results obtained are discussed in the light of the known mutagenic mechanisms of the chemicals tested. We also characterized morphologically transformed foci induced by different chemicals with respect to some biological properties. Anchorage independence was determined by testing growth in soft agar, loss of contact inhibition was quantitated by measuring maximum cell densities and malignancy was tested by tumor induction in nude mice. Although no very close correlation between these parameters and morphology was observed, the most malignant clones are also the ones with the highest values in the other tests. Our data make one or few genetical targets for transformation induction likely. We therefore speculate that the diverse phenotypes obtained might be due to differential activation of one or very few transforming genes in these cells.