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Many stress conditions a cell encounters threaten the continuation of basic biological processes ultimately endangering its survival. Heat shock and antibiotic exposure can lead to a sudden surge of protein un- and misfolding, while nutrient starvation directly causes a lack of energy and molecular building blocks. Our understanding of how cells integrate environmental stress signals, execute protective functions and handle persistent damage is still far from comprehensive. In this thesis the model bacterium Caulobacter crescentus was used to answer basic questions about the regulation and execution of bacterial stress responses and damage clearance. Persistent larger protein aggregates can be maintained as remnants of a past stress exposure and in all of the few bacteria studied to date these particles collect at the poles. In the symmetrically dividing bacterium E. coli this aggregate localization pattern was shown to lead to an old pole lineage-specific retention. In paper I, we studied aggregate formation and inheritance in an asymmetrically dividing bacterium. While aggregates are dissolved by molecular chaperones following moderate heat stress, intense stress induces the emergence of long-lived aggregates. Surprisingly, we find that the majority of persistent aggregates do not collect at the old poles but instead describe a mechanism by which they are constantly displaced towards the new pole. This causes inheritance of aggregates by old and new pole cells at a stable rate without lineage-specific retention, a previously unknown pattern of aggregate inheritance in bacteria. While we found that deletion of most chaperones in C. crescentus does not affect viability in the absence of stress, the mechanistic basis for why DnaK, like in other bacteria, is also required in the absence of stress remains unclear. In paper II, we show that DnaK's function as a negative regulator of the heat shock sigma factor σ32 is essential for viability at physiological temperatures

Many stress conditions a cell encounters threaten the continuation of basic biological processes ultimately endangering its survival. Heat shock and antibiotic exposure can lead to a sudden surge of protein un- and misfolding, while nutrient starvation directly causes a lack of energy and molecular building blocks. Our understanding of how cells integrate environmental stress signals, execute protective functions and handle persistent damage is still far from comprehensive. In this thesis the model bacterium Caulobacter crescentus was used to answer basic questions about the regulation and execution of bacterial stress responses and damage clearance. Persistent larger protein aggregates can be maintained as remnants of a past stress exposure and in all of the few bacteria studied to date these particles collect at the poles. In the symmetrically dividing bacterium E. coli this aggregate localization pattern was shown to lead to an old pole lineage-specific retention. In paper I, we studied aggregate formation and inheritance in an asymmetrically dividing bacterium. While aggregates are dissolved by molecular chaperones following moderate heat stress, intense stress induces the emergence of long-lived aggregates. Surprisingly, we find that the majority of persistent aggregates do not collect at the old poles but instead describe a mechanism by which they are constantly displaced towards the new pole. This causes inheritance of aggregates by old and new pole cells at a stable rate without lineage-specific retention, a previously unknown pattern of aggregate inheritance in bacteria. While we found that deletion of most chaperones in C. crescentus does not affect viability in the absence of stress, the mechanistic basis for why DnaK, like in other bacteria, is also required in the absence of stress remains unclear. In paper II, we show that DnaK's function as a negative regulator of the heat shock sigma factor σ32 is essential for viability at physiological temperatures

Many stress conditions a cell encounters threaten the continuation of basic biological processes ultimately endangering its survival. Heat shock and antibiotic exposure can lead to a sudden surge of protein un- and misfolding, while nutrient starvation directly causes a lack of energy and molecular building blocks. Our understanding of how cells integrate environmental stress signals, execute protective functions and handle persistent damage is still far from comprehensive. In this thesis the model bacterium Caulobacter crescentus was used to answer basic questions about the regulation and execution of bacterial stress responses and damage clearance. Persistent larger protein aggregates can be maintained as remnants of a past stress exposure and in all of the few bacteria studied to date these particles collect at the poles. In the symmetrically dividing bacterium E. coli this aggregate localization pattern was shown to lead to an old pole lineage-specific retention. In paper I, we studied aggregate formation and inheritance in an asymmetrically dividing bacterium. While aggregates are dissolved by molecular chaperones following moderate heat stress, intense stress induces the emergence of long-lived aggregates. Surprisingly, we find that the majority of persistent aggregates do not collect at the old poles but instead describe a mechanism by which they are constantly displaced towards the new pole. This causes inheritance of aggregates by old and new pole cells at a stable rate without lineage-specific retention, a previously unknown pattern of aggregate inheritance in bacteria. While we found that deletion of most chaperones in C. crescentus does not affect viability in the absence of stress, the mechanistic basis for why DnaK, like in other bacteria, is also required in the absence of stress remains unclear. In paper II, we show that DnaK's function as a negative regulator of the heat shock sigma factor σ32 is essential for viability at physiological temperatures

The slow development of effective treatment of glioblastoma is contrasted by the rapidly advancing research on the molecular mechanisms underlying the disease. Amplification and overexpression of receptor tyrosine kinases, particularly EGFR and PDGFRA, are complemented by mutations in the PI3K, RB1, and p53 signaling pathways. In addition to finding effective means to target these pathways, we may take advantage of the recent understanding of the hierarchical structure of tumor cell populations, where the progressive expansion of the tumor relies on a minor subpopulation of glioma stem cells, or gliomainitiating cells. Finding ways to reprogram these cells and block their self-renewal is one of the most important topics for future research.

BackgroundEuploid chromosome balance is vitally important for normal development, but is profoundly changed in many tumors. Is each tumor dependent on its own structurally and numerically changed chromosome complement that has evolved during its development and progression? We have previously shown that normal chromosome 3 transfer into the KH39 renal cell carcinoma line and into the Hone1 nasopharyngeal carcinoma line inhibited their tumorigenicity. The aim of the present study was to distinguish between a qualitative and a quantitative model of this suppression. According to the former, a damaged or deleted tumor suppressor gene would be restored by the transfer of a normal chromosome. If so, suppression would be released only when the corresponding sequences of the exogenous normal chromosome are lost or inactivated. According to the alternative quantitative model, the tumor cell would not tolerate an increased dosage of the relevant gene or segment. If so, either a normal cell derived, or, a tumor derived endogenous segment could be lost.MethodsFluorescence in Situ Hybridization based methods, as well as analysis of polymorphic microsatellite markers were used to follow chromosome 3 constitution changes in monochromosomal hybrids.ResultsIn both tumor lines with introduced supernumerary chromosomes 3, the copy number of 3p21 or the entire 3p tended to fall back to the original level during both in vitro and in vivo growth. An exogenous, normal cell derived, or an endogenous, tumor derived, chromosome segment was lost with similar probability. Identification of the lost versus retained segments showed that the intolerance for increased copy number was particularly strong for 3p14-p21, and weaker for other 3p regions. Gains in copy number were, on the other hand, well tolerated in the long arm and particularly the 3q26-q27 region.ConclusionThe inability of the cell to tolerate an experimentally imposed gain in 3p14-p21 in contrast to the well tolerated gain in 3q26

BackgroundEuploid chromosome balance is vitally important for normal development, but is profoundly changed in many tumors. Is each tumor dependent on its own structurally and numerically changed chromosome complement that has evolved during its development and progression? We have previously shown that normal chromosome 3 transfer into the KH39 renal cell carcinoma line and into the Hone1 nasopharyngeal carcinoma line inhibited their tumorigenicity. The aim of the present study was to distinguish between a qualitative and a quantitative model of this suppression. According to the former, a damaged or deleted tumor suppressor gene would be restored by the transfer of a normal chromosome. If so, suppression would be released only when the corresponding sequences of the exogenous normal chromosome are lost or inactivated. According to the alternative quantitative model, the tumor cell would not tolerate an increased dosage of the relevant gene or segment. If so, either a normal cell derived, or, a tumor derived endogenous segment could be lost.MethodsFluorescence in Situ Hybridization based methods, as well as analysis of polymorphic microsatellite markers were used to follow chromosome 3 constitution changes in monochromosomal hybrids.ResultsIn both tumor lines with introduced supernumerary chromosomes 3, the copy number of 3p21 or the entire 3p tended to fall back to the original level during both in vitro and in vivo growth. An exogenous, normal cell derived, or an endogenous, tumor derived, chromosome segment was lost with similar probability. Identification of the lost versus retained segments showed that the intolerance for increased copy number was particularly strong for 3p14-p21, and weaker for other 3p regions. Gains in copy number were, on the other hand, well tolerated in the long arm and particularly the 3q26-q27 region.ConclusionThe inability of the cell to tolerate an experimentally imposed gain in 3p14-p21 in contrast to the well tolerated gain in 3q26

This project tests the hypothesis that Int6 regulates the 26s proteasome and acts as a tumor suppressor for breast cancer. The authors have accomplished a key step in this study by generating siRNA sequences that knock down Int6 expression, thus influencing mitosis. They also have prepared a good Int6 antibody and will soon learn whether Int6 associates with the proteasome biochemically. They have made great strides in exploring additional angels to better study Int6 functions. First, they have evidence supporting an intriguing possibility that Int6 may be C-terminally truncated in human breast cancer cell lines. Second, their previous studies of the fission yeast system suggest that the tumorigenesis potential induced by Int6 inactivation can be counteracted by oncogenic Ras. They have further investigated the relationship between Int6 and Ras and found that in fission yeast Int6 can co-localize with the Ras-Cdc42 pathway in the endomembrane. They will determine whether this occurs in human cells and ascertain its role in tumorigenesis.

Women found to have atypical hyperplasia on a breast biopsy are at significantly increased risk for breast cancer. Nipple duct lavage (NDL) is being promoted by some as a new screening test for atypical hyperplasia, but cytological interpretation is a subjective art and experience indicates that the underlying conditions represented by cytological atypia on NDL can range from intraductal papilloma to ductal carcinoma in situ (DCIS). Laboratory studies indicate that methylation of tumor suppressor genes is an early event in breast carcinogenesis and often represents a cell's attempt to defeat cell cycle control. We have shown previously that methylation of RASSF1A or APC in benign breast epithelium correlates with calculated breast cancer risk and the finding of Cyclin D2 methylation is specific for malignant transformation. We are applying these objective methylation tests to cells obtained by NDL from women with breast cancer, women at increased risk of breast cancer, and women at low or average risk of breast cancer. Successful completion of the project will provide new tools for the objective evaluation of breast epithelial cells obtained by NDL in order to accurately risk stratify women and to enhance the early detection of DCIS.

In published studies, we have shown that chromosome 11p15.5 exhibits loss of heterozygosity (LOH) in ^60% of breast tumors, and that there is a significant correlation between lip LOH, lymphatic invasion and aggressive metastatic disease. Our data suggests that chromosome 11p15.5 harbors a tumor/metastasis suppressor gene. An intriguing candidate gene that we have mapped to the tumor/metastasis suppressor locus on chromosome 11p15.5 is Integrin-linked Kinase (ILK). ILK is a newly identified ankyrin-repeat containing serine/threonine kinase that binds to the cytoplasmic domains of both 81 and the 83 integrins. Here, we present evidence that the Integrin-Linked Kinase (ILK) gene maps to the commonly deleted chromosome 11p15.5 and suppresses malignant growth of human breast cancer cells both in vitro and in vivo. ILK is expressed in normal breast tissue but-not in metastatic breast cancer cell lines or in advanced breast cancers. Transfection of wild-type ILK into the MDA-MBA35 mammary carcinoma cells potently suppressed their growth and invasiveness in vitro, and reduced the cells' ability to induce tumors and metastasize in athymic mice. Conversely, expression of the ankyrin repeat or catalytic domain mutants of ILK failed to suppress the growth of these cells. Growth suppression by ILK is not due to apoptosis but is mediated by its ability to block cell cycle progression in the G1 phase. These findings directly demonstrate that ILK deficiency facilitates neoplastic growth and suggest a novel role for the ILK gene in tumor suppression., Original contains color plates: All DTIC reproductions will be in black and white.

The bladder is a hollow organ in the small pelvis. It stores urine that is produced when the kidneys filter the blood. Four different layers, the epithelium, lamina propria, muscularis, and connective tissue, define the bladder wall. The epithelium consists of 7 to 10 cell layers and rests on a basal lamina synthesized by epithelial and mesenchymal cells (Figure 1). The thickness of the epithelial cell layer and the lamina propria depends on the degree of distension of the bladder. The cells of the epithelium are referred to as transitional cells. They line the urinary tract starting at the kidney, down the ureter, into the bladder and most of the urethra. Their shape varies between cubical and flat. The latter cells form the barrier between urine and the epithelium. Because of their large fiat morphology these superficial cells are sometimes named umbrella cells. The muscle coat of the bladder is also referred to as the detrusor muscle. It allows the bladder to get larger and smaller as urine is stored or emptied.

We have previously shown that chromosome 11p15.5 exhibits loss of heterozygosity (LOH) in ^60% of breast tumors, and that there is a significant correlation between 11p LOH, lymphatic invasion and aggressive metastatic disease. Our data suggests that chromosome 11p15.5 harbors a metastasis suppressor gene. An intriguing candidate gene that we have mapped to the metastasis suppressor locus on chromosome 11p15.5 is Integrin-linked kinase (ILK). ILK is a newly identified ankyrin-repeat containing serine/threonine kinase that binds to the cytoplasmic domains of both 131 and the 133 integrins., Original contains color plates: All DTIC reproductions will be in black and white.

During the past year our project has evolved dramatically and significant progress has been make with respect to our overall goals of defining the genetic changes that distinguish normal cells from tumor cells of the breast. The most important evolution of the project is our change in strategy from identifying presumed tumor suppressor genes defined by loss of heterozygosity (LOH) using a positional cloning approach, to ascertaining and comparing genetic expression profiles from surgical specimens or cells grown in tissue culture using hybridization of cDNA from our tissues to microarrays of interesting genes. This is a very new technology and we are using a novel micro arraying and scanning instrument recently acquired by our laboratory in collaboration with Molecular Dynamics/Amersham. Preliminary experiments suggest that the sensitivity of our new microarray system is sufficiently high to allow us to detect variance in gene expression levels with high precision even under complex hybridization conditions. We have also applied the Differential Display protocol to identify three genes with altered expression in a model system of normal and neoplastic epithelial colonocytes. As an alternative to determining biologic effects of specific genes, we have tested and selected an amphoteric retroviral conditional expression system, which replaces previous experiments using antisense oligo's. We are confident that the 'modernization' of our research project will facilitate the identification of genes critical to the development and progression of breast cancer.

In our previous annual report, we summarized the accomplishment made towards each of the three specific aims stated in the original grant proposal and we listed the corresponding manuscript and publication generated from those data. Here, we would like to reiterate the objectives of the grant proposal: to characterize the molecular mechanisms by which normal versus tumor breast cells interact with their ECM and the microenvironment, and to determine the functional significance of this interaction and to examine the role of ECM/cell signaling in turmorigenesis. Functions of classical tumor suppressor genes, both Rb and genes on chromosome 11, on the behavior of mammary epithelial cells in 3-D cultures is discussed. Future studies will focus on implementing molecular biology approaches to provide fundamental clues to the mechanisms underlying the breast tumor development., Original contains color plates; All DTIC/NTIS reproductions will be in black and white.

By a three-dimensional reconstituted basement membrane (EHS matrix) assay, we have previously shown that breast morphogenesis is regulated by cell-extracellular matrix (ECM) interactions and these contacts are impaired in malignancy. A series of studies were conducted to investigate the significance of cell/ECM interactions in tumorigenesis. ECM influences expression of lactoferrin, a differentiation marker, either by altering cell-cell interactions or by providing mechanochemical signals that regulate cell shape. Emphasis on the importance of intact beta-1-integrin signaling was indicated by function blocking studies. Phenotypically normal HMT-3522 cells failed to grow or differentiate and underwent apoptosis in the presence of beta-1 integrin blocking antibodies. In contrast a number of tumorigenic cell lines demonstrated refractoriness to these effects indicating they have circumvented this regulatory pathway. Finally the importance of cell/ECM interactions in the maintenance of the differentiated phenotype was underscored by examining the effects of overexpression of the putative tumor suppressor gene nm23-H1 in metastatic cell line MDA-MB- 435, where several aspects of normal breast morphogenesis was recapitulated. Further studies are presently underway to dissect other ECM components and their signaling pathways that are critical in breast cancer progression.

It is nowadays generally accepted that cancer can be considered as a genetic disease. However, there are two clear differences between cancer and most other genetic diseases. First, most cancers are caused essentially by somatic mutations, whereas all other genetic diseases are caused solely by germline mutations. Second, for most cancer types at least two mutations are required before a tumor may arise. For most pediatric and some adult tumors only a limited number of mutations are supposed to be sufficient for tumor development (Knudson, 1971, 1986; Haber and Housman, 1992). In contrast, most adult cancers appear to require more than two genetic changes. This 'multiple-hit' concept of tumorigenesis stems from several lines of evidence. First, most adult cancers show an exponential increase in incidence with age, suggesting that the accumulation of different hits is involved in tumor development (Vogelstein and Kinzler, 1993). Second, a dramatically increased incidence of cancer has been observed in individuals with chromosome instability syndromes such as Bloom's syndrome (Vijayalaxmi et aI., 1983; Seshadri et aI., 1987). Third, repeated administration of mutagen to test animals is generally required before a tumor arises (Saffhill et aI., 1985). Fourth, increasing evidence becomes available for the involvement of multiple genetic alterations in common human tumors. The most extensively studied example is the accumulation of genetic alterations observed in human colon tumors, in which mutations in APe and ras found in adenomatous polyps have been classified as early events and mutations in p53 and Dee as later events that may contribute to tumor progression (Fearon and Vogelstein, 1990; section 3.3). In a specific human cancer, mutations in one particular gene appear to precede those in others (Vogelstein and Kinzler, 1993), although after this first hit the accumulation of changes rather than their specific order seems to be important for tumor progression (Marx

© 2018 Chulpanova, Kitaeva, Tazetdinova, James, Rizvanov and Solovyeva. Mesenchymal stem cells (MSCs) are non-hematopoietic progenitor cells, which can be isolated from different types of tissues including bone marrow, adipose tissue, tooth pulp, and placenta/umbilical cord blood. There isolation from adult tissues circumvents the ethical concerns of working with embryonic or fetal stem cells, whilst still providing cells capable of differentiating into various cell lineages, such as adipocytes, osteocytes and chondrocytes. An important feature of MSCs is the low immunogenicity due to the lack of co-stimulatory molecules expression, meaning there is no need for immunosuppression during allogenic transplantation. The tropism of MSCs to damaged tissues and tumor sites makes them a promising vector for therapeutic agent delivery to tumors and metastatic niches. MSCs can be genetically modified by virus vectors to encode tumor suppressor genes, immunomodulating cytokines and their combinations, other therapeutic approaches include MSCs priming/loading with chemotherapeutic drugs or nanoparticles. MSCs derived membrane microvesicles (MVs), which play an important role in intercellular communication, are also considered as a new therapeutic agent and drug delivery vector. Recruited by the tumor, MSCs can exhibit both pro- and anti-oncogenic properties. In this regard, for the development of new methods for cancer therapy using MSCs, a deeper understanding of the molecular and cellular interactions between MSCs and the tumor microenvironment is necessary. In this review, we discuss MSC and tumor interaction mechanisms and review the new therapeutic strategies using MSCs and MSCs derived MVs for cancer treatment.