Expression Of Cellular Proto-oncogenes Research Articles

Insertional Mutagenesis by Retroviral Vectors: Current Concepts and Methods of Analysis

Retroviral vectors derived from gammaretroviruses or lentiviruses have now been used extensively in clinical gene therapy trials for several diseases including primary immunodeficiencies, beta thalassaemia and adrenoleukodystrophy. Their utility in this setting has been readily demonstrated by the largely favourable outcomes in recent clinical trials, however this success has been marred by the emergence of malignancies in some trials. These malignancies were a consequence of perturbation of cellular proto-oncogene expression by the integrated retroviral vectors, the process of which is referred to as 'insertional mutagenesis' (IM). In this review, the origins of our understanding of IM are reviewed and applied to the clinical gene therapy trials conducted with retroviral vectors. Old and new methods for assessing this phenomenon are discussed with a view to provide a comprehensive account of this emerging field.

Genetics of mouse mammary tumor virus-induced mammary tumors: linkage of tumor induction to the gag gene.

Retroviruses are believed to induce tumors by acting as insertional mutagens that activate expression of cellular protooncogenes. Indeed, almost 90% of mouse mammary tumor virus (MMTV)-induced mammary tumors in C3H/He mice show upregulation of Int protooncogenes. We have analyzed three different MMTV variants [MMTV(C3H), MMTV(HeJ), and a genetically engineered MMTV hybrid provirus (HP)] for tumorigenicity in mice from two distinct genetic backgrounds. All three viruses were tumor causing in BALB/cJ mice. However, only MMTV(C3H), but not MMTV(HeJ) or HP, induced mammary tumors in C3H/He mice. All of the viruses were infectious on either background and up-regulated expression of Int genes in tumors they induced. Like HP, MMTV(HeJ) was found to be a genetic recombinant between endogenous Mtv1 provirus and exogenous MMTV(C3H). Sequence comparison of MMTV variants linked the tumorigenicity of MMTV(C3H) to the gag region of the retrovirus.

Progesterone Pretreatment Inhibits the Expression of c-ski mRNA and Epithelial Cell Proliferation Induced by Estrogen in the Rat Uterus

Progesterone has been shown to inhibit the proliferative action of estrogen in the uterine epithelial cells. We have previously reported that the expression of cellular protooncogene, c-ski, is induced by estrogen treatment in the uterine epithelial cells of ovariectomized rats, and suggested its role in the proliferation of uterine epithelial cells. In the present study, we examined whether progesterone pretreatment would affect the estrogen-induced expression of c-ski mRNA in the uterine epithelial cells. In situ hybridization analysis revealed that estrogen-induced expression of c-ski mRNA was attenuated by progesterone pretreatment in the uterine luminal epithelial cells, accompanied by the decrease in the number of proliferating cell nuclear antigen-positive cells among the luminal epithelial cells. The results obtained further support our hypothesis that c-ski plays a role in proliferation of uterine epithelial cells and mediates the proliferative action of estrogen.

Endometrial expression of cellular protooncogene c- ski and its regulation by estradiol-17β

The expression of the cellular protooncogene c- ski was examined in the rat uterus. In situ hybridization revealed that c- ski mRNA was expressed in the uterus of the adult rat on the day of estrous and localized mainly in the luminal and glandular epithelia. To test the possibility that the expression of c- ski mRNA is induced by estrogen, rats were ovariectomized and estradiol-17β (E 2) was injected. The expression of c- ski mRNA was upregulated 3 h after E 2 treatment, reaching the highest level at 6 h and this persisted until 24 h; the E 2-induced expression of c- ski mRNA was restricted to the luminal and glandular epithelia. These results suggest that the c- ski gene plays a role in uterine epithelial cell proliferation and mediates the proliferative action of E 2.

Molecular Genetic Alterations of c-myc Oncogene in Superficial and Locally Advanced Bladder Cancer

Objective: Gene activation and altered expression of cellular proto-oncogene are important mechanisms implicated in initiation and development processes of human cancer. It has already been shown that c-myc oncogene is implicated in the control of cell proliferation, apoptosis and differentiation. Methods: We have determined the methylation status, the presence of genetic amplification and the presence of m-RNA overexpression of c-myc gene in 31 samples from patients with bladder carcinomas. Results: Our data demonstrated the presence of c-myc gene amplification only in 5 of 15 superficial bladder carcinomas (p < 0.05). On the other hand, we did not find statistical significant correlation between the methylation, expression of c-myc gene and the clinical-histopathological parameters. A significant correlation (p < 0.05) was found between the methylation pattern and m-RNA overexpression of c-myc oncogene. Conclusion: We demonstrate aberrant c-myc gene status in human bladder cancer. This oncogene is altered at different levels in bladder carcinoma genesis and progression.

Meis1, a PBX1-Related Homeobox Gene Involved in Myeloid Leukemia in BXH-2 Mice

Leukemia results from the accumulation of multiple genetic alterations that disrupt the control mechanisms of normal growth and differentiation. The use of inbred mouse strains that develop leukemia has greatly facilitated the identification of genes that contribute to the neoplastic transformation of hematopoietic cells. BXH-2 mice develop myeloid leukemia as a result of the expression of an ecotropic murine leukemia virus that acts as an insertional mutagen to alter the expression of cellular proto-oncogenes. We report the isolation of a new locus, Meis1, that serves as a site of viral integration in 15% of the tumors arising in BXH-2 mice. Meis1 was mapped to a distinct location on proximal mouse chromosome 11, suggesting that it represents a novel locus. Analysis of somatic cell hybrids segregating human chromosomes allowed localization of MEIS1 to human chromosome 2p23-p12, in a region known to contain translocations found in human leukemias. Northern (RNA) blot analysis demonstrated that a Meis1 probe detected a 3.8-kb mRNA present in all BXH-2 tumors, whereas tumors containing integrations at the Meis1 locus expressed an additional truncated transcript. A Meis1 cDNA clone that encoded a novel member of the homeobox gene family was identified. The homeodomain of Meis1 is most closely related to those of the PBX/exd family of homeobox protein-encoding genes, suggesting that Meis1 functions in a similar fashion by cooperative binding to a distinct subset of HOX proteins. Collectively, these results indicate that altered expression of the homeobox gene Meis1 may be one of the events that lead to tumor formation in BXH-2 mice.

Analysis of retrotransposon families in genomic DNA by two-dimensional restriction mapping: Detection of VL30 insertions in mouse thymic lymphoma

Reinsertion of mammalian retrotransposable genetic elements is known to be causally associated with tumourigenesis, typically through mechanisms involving insertional deregulation of cellular protooncogene expression. We report here on the application of a two-dimensional restriction mapping-Southern hybridisation approach for analysis of retrotransposon families of low to moderate genetic complexity, which is particularly suited to pairwise comparisons between DNA samples. By using this method, non-constitutional mink-cell-focus-forming type retro-elements were readily detectable in AKR mouse thymic lymphomas against a background of approx. 30 related elements in control DNA. However, in the WEHI 3B myeloid leukaemia cell line, the resolution of two-dimensional mapping permitted detection of only occasional reinsertions of intracisternal A particle retro-elements (genetic complexity: 10 3). In analysing the VL30 family of retrotransposon (genetic complexity: 150) we developed a strategy for identifying the known transcriptionally active sub-set of these elements in genomic DNA through the generation of an internal, diagnostic restriction fragment. Moreover, in some cases of thymic lymphoma, several candidate re-insertions of VL30 elements were detected, consistent with a suggested role for retrotransposition of this class of element in lymphomagenesis of retroviral aetiology.

Phenomena of Spontaneous Proliferation, Differentiation, and Apoptosis in Primary Culture of Leukemic Lymphocytes

The proliferative capacity of lymphocytes from peripheral blood of bovine with chronic lymphocytic leukemia (CLL) in vitro was investigated. We have shown earlier that CLL cells spontaneously proliferate in serum-free medium in the absence of added growth factors and mitogenic stimulation; autocrine growth factors provide the growth-initiating signal for CLL cells. The results of the present study showed that bovine serum albumin or fetal calf serum greatly enhanced the number of CLL cells incorporating [3H]thymidine. Although some CLL cells proceeded through more than one cell cycle, proliferation of CLL cells in culture was temporary. On the other hand, it was shown that CLL cells differentiated spontaneously in culture. This differentiation was characterized by the appearance of plasmacytoid cells possessing cytoplasmic immunoglobulins that coincided with the cessation of cell proliferation. Moreover, together with spontaneous proliferation and differentiation, the phenomenon of programmed cell death (apoptosis) was found, as was evidenced by the appearance of apoptotic bodies as well as DNA fragmentation. The findings indicate that the loss of proliferative potential of CLL cells in culture may be a consequence of their differentiation and/or apoptosis in vitro. CLL cells, with an autotrine growth mechanism, spontaneous differentiation, and apoptosis in vitro, provide a new model system for studies of the relationship between cellular proto-oncogene expression and inhibition of growth and/or induction of differentiation.

Cellular Proto-Oncogene Expression Following Exposure of Mice to γ Rays

Many studies have shown the importance of altered cellular proto-oncogene expression in contributing to changes in cell survival, cell transformation, and cell cycle progression. In these experiments we examined the effects of total-body exposure of BCF1 mice to gamma rays (3 Gy) in modulating expression of cellular oncogenes in both gut and liver tissues. We selected specific cellular oncogenes (c-fos, c-myc, c-src, and c-H-ras), based on their normal expression in liver and gut tissues from untreated mice. As early as 5 min following whole-body exposure of BCF1 mice to gamma rays we detected induction of mRNA specific for c-src and c-H-ras in both liver and gut tissues. Accumulation of c-fos-RNA was slightly decreased in gut but was unaffected in liver tissue from irradiated mice relative to untreated controls. Accumulation of c-myc mRNA was unaffected in all tissues examined. These experiments document that modulation of cellular proto-oncogene expression can occur as an early event in tissues following irradiation and suggest that this modulation may play a role in radiation-induced cellular changes.

Anchorage-independent growth and the expression of cellular proto-oncogenes in normal human epidermal keratinocytes and in human squamous cell carcinoma cell lines

The expression of multiple cellular proto-oncogenes and the in vitro anchorage-independent growth of normal human epidermal keratinocytes and several human squamous cell carcinoma cell lines were studied and correlated. Squamous cell carcinoma cell lines KB, Si Ha, HEp-2, and Fa Du showed high anchorage independency, and MS 751 and A-253 cell lines had minimum independency. However, the normal keratinocytes and the A-431 cell line did not show anchorage-independent growth. Both the normal human epidermal keratinocytes and cancer cell lines expressed multiple proto-oncogenes such as src, erb B-1, abl, fos, raf, H- ras, and myc, and the amount of expression of these oncogenes was notably higher in the cancer cell lines than in the normal keratinocytes. The expression of proto-oncogenes from the monolayer cultures of the cancer cell lines is poorly correlated with the anchorage independency of the cells. These data indicate that the anchorage independency is not directly linked to the expression of specific cellular proto-oncogene(s) of the monolayer cancer cell cultures.

DNA integration sites and hepatocellular carcinoma

This study is part of an ongoing analysis of woodchuck hepatitis virus integration sites in the host genome of hepatocellular carcinomas. The study of woodchuck hepatitis virus-DNA integration sites may shed light on the oncogenic mechanisms involved in cellular transformation and tumor formation. Viral integration enhancing cellular proto-oncogene expression is one such mechanism and has been well documented for oncogenic retroviruses such as mouse mammary tumor virus and interleukin-1. By cloning a woodchuck hepatitis virus integration site from a woodchuck hepatocellular carcinoma the authors were able to identify a new member of the myc gene family, N-myc-2. Examination of 30 additional woodchuck hepatomas revealed viral integration commonly occurred near N-myc loci with an additional five woodchuck hepatitis virus integrants near the N-myc-2 gene and one viral integrant near N-myc-1. Three of these N-myc-2 viral integrations were further evaluated and found to be localized within 200 bp of the translation stop codon. This 3′ noncoding region has recently been identified as a common site of murine leukemia virus integration in virally induced T-cell lymphomas and results in increased expression of the N-myc gene. Similar mechanisms can be proposed for hepatocellular carcinoma formation. Woodchuck hepatitis virus integration near cell-growth related protooncogenes, such as N-myc, can juxtapose viral enhancer elements and growth-regulatory genes. Virally induced overexpression of proto-oncogene messenger RNA could result from enhanced transcription or increased messenger RNA stability. To search for such effects the authors analyzed N-myc-2 RNA levels in 30 woodchuck hepatitis virus-related hepatomas. Increased levels of N-myc-2 RNA were found in 18 of 30 tumors, whereas nontumorous portions of the same livers had no detectable N-myc-2 RNA. Taken together these findings suggest that woodchuck hepatitis virus integration can result in altered N-myc-2 gene expression in a significant proportion of woodchuck hepatocellular carcinomas. The deregulation of N-myc gene expression could result in cellular transformation and ultimately tumor formation. Such examples of hepadnavirus-specific oncogenic mechanisms lend credence to theories of hepatitis B virus-induced tumorigenesis and provide models to design molecular investigations of human hepatocellular carcinoma formation.

Sequential protooncogene expression during regeneration in rat stomach

Cellular protooncogenes such as c-myc and c-Ha-ras may play important roles in the control of regeneration of the stomach. In this study, in situ hybridization histochemistry and immunohistochemistry were used to determine how these protooncogenes and the corresponding oncoproteins are expressed at the cellular level during gastric regeneration after mucosal injuries caused by indomethacin. In addition, cells in the S-phase were immunohistochemically detected by means of 5-bromo-2′-deoxyuridine incorporation. Expression of the c-myc gene was localized to nuclei and reached a maximum at 3 h, and that of the c-Ha-ras gene was localized to cytoplasm with a peak at 6–12 h after treatment on the mucous neck, parietal, chief, and enterochromaffinlike cells around the lesions. The distribution of cells in the S-phase roughly coincided with that of cells in which expression of the protooncogenes was detected. In conclusion, various types of gastric mucosal cells participated in the sequential regulated expression of cellular protooncogenes during regeneration of the rat stomach.

Rapid induction of polyadenylated H1 histone mRNAs in mouse erythroleukemia cells is regulated by c-myc.

Chemically induced differentiation of murine erythroleukemia cells is a multistep process involving a precommitment period in which exposure to inducer leads to cells that are irreversibly committed to terminal differentiation. Certain changes in the expression of cellular proto-oncogenes are an important feature of the precommitment phase. We have identified two H1 histone genes that are rapidly induced during this period. Unlike most histone genes, these two H1 genes encode polyadenylated mRNAs with long 3' untranslated regions. To investigate the relationship between induction of the H1 mRNAs and changes in proto-oncogene expression, we studied two independent series of mouse erythroleukemia cell lines that are inhibited from differentiating because of deregulated expression of transfected copies of c-myc or c-myb. The results showed that induction of the H1 mRNAs was negatively regulated by c-myc. The two H1 histone genes are among the first examples of specific cellular genes that are regulated by c-myc. The timing of their induction suggests that they may play an important role in achieving commitment to terminal differentiation.

Rapid induction of polyadenylated H1 histone mRNAs in mouse erythroleukemia cells is regulated by c-myc.

Chemically induced differentiation of murine erythroleukemia cells is a multistep process involving a precommitment period in which exposure to inducer leads to cells that are irreversibly committed to terminal differentiation. Certain changes in the expression of cellular proto-oncogenes are an important feature of the precommitment phase. We have identified two H1 histone genes that are rapidly induced during this period. Unlike most histone genes, these two H1 genes encode polyadenylated mRNAs with long 3' untranslated regions. To investigate the relationship between induction of the H1 mRNAs and changes in proto-oncogene expression, we studied two independent series of mouse erythroleukemia cell lines that are inhibited from differentiating because of deregulated expression of transfected copies of c-myc or c-myb. The results showed that induction of the H1 mRNAs was negatively regulated by c-myc. The two H1 histone genes are among the first examples of specific cellular genes that are regulated by c-myc. The timing of their induction suggests that they may play an important role in achieving commitment to terminal differentiation.

C-myc protooncogene expression in mouse erythroleukemia cells.

Murine erythroleukemia (MEL) cells are erythroid progenitors whose programs of erythroid differentiation has been interrupted by transformation with the Friend virus complex. As a result of the ability of certain chemicals such as dimethylsulfoxide (DMSO) to induce terminal erythroid differentiation, the cells have been used as a model for understanding the molecular basis of cellular differentiation. Recent work on MEL cells as well as other differentiating systems indicates that expression of cellular protooncogenes is implicated in chemically mediated differentiation. In MEL cells the expression of the c-myc protooncogene undergoes unusual biphasic changes following inducer treatment. Levels of c-myc mRNA decrease 10- to 20-fold between 1 and 2 hr and are then reexpressed between 12 and 24 hr. These changes occur as a result of complex transcriptional and posttranscriptional regulatory events. Recent DNA transfection experiments, in which MEL cells were transfected with myc expression vectors, indicate that both the early decrease in c-myc expression and its subsequent reexpression are important events in the differentiation pathway. The work on MEL cells, as well as on other models of differentiation, is directed at understanding the molecular basis of leukemogenic transformation and cellular differentiation. The ability of c-myc, as well as other protooncogenes, to influence both of these events indicates that cellular protooncogenes play a central role in their regulation.

Sequential expression and cooperative interaction of c-Ha-ras and c-erbB genes in in vivo chemical carcinogenesis.

The level of expression of several cellular protooncogenes is examined at different stages of 7,12-dimethylbenzanthracene (DMBA)-induced tumor development in hamster buccal pouch epithelium (HBPE). Results presented demonstrate overexpression of c-Ha-ras gene at a very early stage of tumor development, and this elevated level of expression of the gene persists throughout the tumorigenesis process. The expression of the cellular protooncogene c-erbB, on the other hand, can be detected only after 8-10 weeks of DMBA treatment of the tissue and increases with the progression of the disease. The overexpression of c-erbB gene can be correlated with the stage of extensive proliferation and subsequent invasion of the HBPE cells into the underlying connective tissue. This sequential pattern of stage-specific expression of the two cellular protooncogenes can be observed in (i) treated tissues, (ii) stage-representative cultured cells, and (iii) NIH 3T3 transformants derived with DNA from HBPE cells. The low-level expression of c-myc and c-sis genes detected in control tissues remains unaffected, while c-fos gene activity cannot be detected at any stage of tumor development. The overexpression of c-Ha-ras gene alone in HBPE cells derived from tissues treated for 5 weeks (DM5) is not sufficient to induce tumors in athymic mice, whereas expression of c-Ha-ras and c-erbB genes at later stages of tumor development (DM10 and HCPC cells) induce histopathologically defined epithelial cell carcinoma in athymic mice within 2-3 weeks. The sequential overexpression of c-Ha-ras and c-erbB genes in a stage-specific manner and their cooperative interaction in the DMBA-induced in vivo oral carcinogenesis have been demonstrated.

Site-Selective Cyclic AMP Analogs as New Biological Tools in Growth Control, Differentiation, and Proto-oncogene Regulation

The physiologic role of cyclic adenosine monophosphate (cAMP) in the growth control of a spectrum of human cancer lines, including leukemic lines, and v-rasH oncogene-transformed NIH/3T3 cells is demonstrated by the use of site-selective cAMP analogs. These cAMP analogs, which can select either of the two known cAMP binding sites of the cAMP receptor protein, induce potent growth inhibition, phenotypic change, and differentiation (leukemic cells) of cancer cells at micromolar concentrations with no sign of cytotoxicity. The growth inhibition parallels selective modulation of cAMP-dependent protein kinase isozymes, type I versus type II, and suppression of cellular proto-oncogene expression. Site-selective cAMP analogs thus provide new biological tools for investigating cell proliferation and differentiation and also for the improved management of human cancers.

Steroid-induced cell proliferation in vivo is associated with increased c-myc proto-oncogene transcript abundance.

Enhanced c-myc transcript abundance has been observed in a variety of human malignancies, in normal liver tissue induced to proliferate in vivo by partial hepatectomy and in cells in culture induced to proliferate with the addition of protein hormones and growth factors. Little is known, however, about the expression of cellular proto-oncogenes in cells induced to proliferate in vivo by steroid hormones. Experiments reported here indicate that when cells of the immature chicken oviduct are induced to undergo rapid in vivo proliferation by application of the estrogen hormone 17 beta-estradiol, the onset of this proliferation is associated with a rapid, large, and transient increase in c-myc transcript abundance. When estrogen is administered to chickens in which the oviduct has already differentiated, neither massive cell proliferation nor large increases in c-myc transcript abundance are induced. We conclude that the abundance of c-myc transcripts in vivo correlates well with the degree of cell proliferation induced by steroid hormone.

Estrogen induces expression of c-fos and c-myc protooncogenes in rat uterus.

Estrogen stimulates DNA synthesis and cell proliferation in the luminal and glandular epithelia of rodent uterus. We tested the hypothesis that the mitogenic effect of estrogen occurs via activation of the expression of cellular proto-oncogenes by measuring the rate of transcription of 20 proto-oncogenes (abl, bas, erb-A, erb-B, ets, fms, fos, fps/fes, mos, myb, myc, N-myc, raf, Ha-ras, Ki-ras, N-ras, rel, sis, src, and B-lym) in the uterus of ovariectomized rats before and after injection of estrogen. c-onc transcriptional activity was monitored both by an in vitro transcription assay on isolated nuclei (run-on) and by analysis of mature mRNA. c-fos and c-myc proto-oncogenes were found to respond to estrogen with increased expression: c-fos within 30 min, with a first, sharp peak at 2 h and c-myc within 1.5 h, with a first, broad peak at 4-6 h. DNA synthesis start to increase in the uterus 13 h after estrogen injection and show a first peak at 24 h. In the liver and muscle of the same animals there is neither elevation of c-fos and c-myc expression nor increase of DNA synthesis. The kinetics of the induction by estrogen of c-fos gene expression in the uterus parallels the rate of formation of active nuclear estrogen-receptor complex. Furthermore, the ability of estrogen to induce c-fos mRNA was not abolished by the protein synthesis inhibitor cycloheximide.(ABSTRACT TRUNCATED AT 250 WORDS)

Activation of Na+/H+ exchange and the expression of cellular proto-oncogenes in mitogen- and phorbol ester-treated lymphocytes.

It has been suggested that an intracellular alkalinization, resulting from stimulation of Na+/H+ exchange, is a necessary step and perhaps the signal leading to cellular proliferation in cells stimulated by mitogens. This hypothesis was tested by measuring the early stages of the proliferative cascade in cells where antiport activity was precluded by omission of Na+ or by the addition of potent amiloride analogs. To circumvent possible nonspecific effects due to long incubations under these conditions, an early response to mitogens, the increased level of c-fos mRNA, was monitored. In rat thymic lymphocytes, the increase in the level of c-fos RNA induced by the combination of 12-O-tetradecanoylphorbol 13-acetate and ionomycin was unaffected by inhibition of the antiport with 5-(N-ethyl-N-propyl)amiloride. Increased c-fos RNA was also observed in the absence of Na+ and when alkalinization was prevented by means of nigericin. Similar results were obtained with phytohemagglutinin-stimulated human T lymphocytes. Moreover, although the lectin stimulated the antiport in these cells, an alkalinization was not observed, due to the concomitant occurrence of an acidifying process. It was concluded that the stimulation of the Na+/H+ antiport that accompanies the addition of mitogens is neither sufficient nor necessary for the initiation of cellular proliferation.