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The process of oncogenic transformation in vitro has been examined in an attempt to define the molecular mechanisms of carcinogenesis. Transformation in Ki-MSV-infected rat adrenal cortex cells appears to be a multistep process (Auersperg et al., 1981), as does the process of transformation in other nonestablished cells (Land et at., 1983b). The Ki-MSV-infected adrenal cortex cells initially express a partially transformed phenotype and after further passaging progress to a highly transformed phenotype (Calderwood and Auersperg, 1984). Examination of Ki-MSV-infected adrenal cortex cells indicated that progression to a highly transformed phenotype could occur in the absence of significant changes in the level of the expression of the viral ras oncogene. These results indicated that an oncogenically activated ras gene could be expressed in these nonestablished cells in the absence of transformation. Since ras and myc cooperate to transform primary fibroblasts the effect of the co-introduction of myc on Ki-MSV-induced transformation of adrenal cortex cells was examined. It could be demonstrated that myc and ras cooperate to transform the adrenal cortex cells more efficiently than either oncogene alone, but that the infected cultures initially only express some of the phenotypes associated with transformation. The appearance of a fully transformed phenotype, as monitored by growth in soft agar, was not expressed until several passages after infection. An analysis of the Ki-MSV/MMCV-infected cultures indicated that some of the phenotypes associated with activated oncogenes in immortalized cell lines appeared to be suppressed in the coinfected adrenal cortex cells. Transformation by ras and myc appears to require a further cellular change resulting in a loss of the suppression of oncogene action. The emergence of transformed cultures from the Ki-MSV-infected rat adrenal cortex cells was correlated with the reduced expression of a novel ras-related protein of 27000 Mr. Transformation induced by src and myc was also examined. These two oncogenes appeared to cooperate in a two step pathway of transformation that was not susceptible to cellular suppression. The transformed phenotype did not appear to be entirely free of external influence as the growth rate of the transformed cells could be modified by culture conditions. The ability of myc to cooperate with src and ras in the transformation of the early passage adrenal cortex cells provides further support for mutistep carcinogenesis. The effect of oncogenes on steroidogenesis was examined in the Y-1 adrenocortical tumour cell line. The effect of the virally borne oncogenes on growth and morphology of the Y-1 cells was relatively subtle. The oncogenes appear to stimulate the production of fluorogenic steroids, each in a distinct fashion. A model of transformation can be derived in which the roles of the oncogenes and their interaction with the cell can be evaluated. The differences in the pathways of transformation for the two combinations of oncogenes illustrates the potential complexity of the transformation process and provides an in vitro model system for further study. / Science, Faculty of / Microbiology and Immunology, Department of / Graduate

The bovine papillomaviruses (BPV) are capable of transforming cells from a wide range of species both in vivo and in vitro. The 69% portion of the BPV-1 viral genome that contains the transforming region, as well as the 31% portion, were cloned in the pBR322 plasmid vector. An extensive restriction endonuclease map of the transforming region was prepared. Using the cloned transforming region as a ³²P probe, BPV-1 coded mRNA transcripts from transformed cells were detected and sized using the northern blot technique. The largest open reading frame of the transforming region of the BPV-1 genome was sequenced using the Maxam and Gilbert chemical method and the amino acid sequence of a protein that this region could code for was presented.

The papillomaviruses do not appear to be capable of passage in cultured cell lines despite numerous attempts to identify conditions permissive for their propagation. These viruses induce benign tumors in animals (warts, papillomas) by unknown mechanisms. A broad range of animal species is susceptible to papillomavirus-induced tumorogenesis. The basic molecular mechanisms of papillomavirus replication, transcription, and translation to produce virus-specific products in cells are unknown. Since the viruses do not reproduce in vitro, no conditional-lethal and deletion mutants like those characterized in other systems exist. Thus, progress in understanding the papillomavirus-host cell interactions leading to neoplasia has been severely hindered. This dissertation is concerned with biochemical analysis of bovine papillomavirus type 1 (BPV-1) DNA and RNA in BPV-1 transformed and tumor cells. The specific conditions of infection resulting in stable lines of BPV-1 transformed cells are described. Colonies of BPV-1 transformed cells exhibiting anchorage-independent growth in agarose-containing medium were isolated and cloned cell lines were established. Formation of tumors in a rabbit following inoculation with BPV-1 is reported and represents the first evidence of BPV-induced tumorogenesis in this animal species. The BPV-1 transformed and tumor cells are characterized with respect to the quantity and physical state of the BPV-1 genome in the cells. The BPV-1 DNA is present in high copy numbers in free, non-integrated supercoiled and nicked open-circular forms. No evidence of integrated BPV-1 sequences is noted. This is unusual since all other characterized DNA tumor viruses require covalent integration of at least a portion of the viral DNA in the cellular genome during transformation. A detailed restriction endonuclease cleavage map of the BPV-1 genome is presented, representing a more complete physical characterization of the viral genome. The first evidence of BPV-1 specific transcripts in transformed cells is reported. These results should aid in functional characterizations of the BPV-1 genome, particularly the determination of the specific regions of the BPV-1 DNA transcribed during transformation of cells, analogous to early regions defined in other DNA tumor virus systems, and analysis of post-transcriptional processing mechanisms involved in the synthesis of BPV-1 specific messenger RNA.

No description available.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 6 uppsatser.

Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 5 uppsatser.

Thesis (Ph. D.)--University of Texas at Austin, 2000. / Vita. Includes bibliographical references (leaves 168-188). Available also in a digital version from Dissertation Abstracts.

Tang, Ho Lam. / "December 2010." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 119-132). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.

In the normal adult human, hemopoiesis appears to be maintained by the simultaneous activity of many stem cell-derived clones. Conversely, most examples of human myeloid malignancies have been shown to represent clonal populations arising as a result of the unregulated expansion of a single transformed hemopoietic stem cell. The limits of the proliferative capacity of normal hemopoietic stem cells in humans and their persistence in hemopoietic malignancies have, however, not been extensively Investigated. One of the most likely reasons for this is the lack, until very recently, of a widely applicable method to analyze the clonality status of human cell populations. Methylation analysis of two polymorphic genes. HPRT and PGK, now allows such studies to be performed in approximately 50 % of females. The possibility that normal human hemopoietic stem cells might have the capacity to mimic the behaviour of some transformed stem cells by generating clones of progeny that could dominate the entire hemopoietic system was then examined. Such a phenomenon has been well documented in animal models of marrow cell transplantation. I therefore undertook an analysis of all allogeneic marrow transplants performed over a 1 to 1-1/2 year period where the genotype of the donor made clonality analysis using the HPRT or PGK systems possible. Using this approach, I obtained evidence in two patients suggesting that a single or, at most, a very small number of normal primitive hemopoietic stem cells were able to reconstitute the hemopoietic system. In one case the data suggested that such reconstitution was likely to have derived from a stem cell with both lymphopoietic and myelopoietic potential. However, in all other cases hemopoiesis in the transplant recipient was found to be polyclonal. Such findings indicate that clonal dominance in the hemopoietic system is not sufficient to infer that a genetically determined neoplastic change has occurred. In addition, these findings have implications for the design of future gene therapy protocols. The same methodology was also applied to investigate the clonality of different hemopoietic cell populations in patients with chronic myelogenous leukemia (CML) and essential thrombocytosis (ET). In both of these myeloproliferative disorders, the neoplastic clone produces terminally differentiated progeny that appear minimally different from normal. Data from the CML studies confirmed the non-clonal nature of the cells emerging in long-term CML marrow cultures. Similarly, patients transplanted with cultured autologous marrow were shown to undergo polyclonal and bcr-negative reconstitution of their hemopoietic system. Analysis of a series of patients with a clinical diagnosis of ET showed that polyclonal hemopoiesis in the presence of an amplified neoplastic clone is not a rare event in this disorder, and that clonality results do not always correlate with other neoplastic markers associated with myeloproliferative diseases in general. Another example of polyclonal hemopoiesis in the presence of an amplified neoplastic clone was demonstrated in a patient with Ph¹-positive ALL whose disease appeared to have originated in a lymphoid-restricted stem cell. The studies described in this thesis reveal a level of complexity of normal and neoplastic stem cell dynamics not previously documented. They highlight the need for more precise information about the molecular basis of regulatory mechanisms that govern hemopoietic cell proliferation and survival at every level of differentiation. Finally they support the accumulating evidence that acquisition of full malignant potential requires several additive genetic changes first postulated many years ago as the somatic mutation theory of carcinogenesis. / Medicine, Faculty of / Pathology and Laboratory Medicine, Department of / Graduate