Department of Laboratory Medicine and Pathology Faculty Papers and Data

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Development of blood assays for multiple system atrophy and Parkinson disease
(2025-09-30) Shi, Min
Clinical diagnosis and differential diagnosis of Parkinson disease (PD) and multiple system atrophy (MSA), two progressive and fatal neurodegenerative disorders, can be quite challenging, especially at early disease stages, due to the overlapping clinical symptoms among various parkinsonian disorders. Biomarkers such as neuroimaging measurements and α-syn seeding aggregation assays (Real-Time Quaking-Induced Conversion or RT-QuIC) in cerebrospinal fluid (CSF) and some biopsy samples have shown promising results in improving PD and MSA diagnosis, but simpler, less expensive, accurate, and reliable biochemical markers, particularly those in easily accessible body fluids (e.g., blood), are still urgently needed to aid in clinical assessments. Growing evidence suggests that membrane-bound extracellular vesicles (EVs) play important roles in cell-to-cell communication and signaling and the pathology of neurodegenerative diseases, including PD and MSA. Additionally, EVs carrying unique disease-specific and functionally important cargo may cross the blood-brain barrier and be detected in vivo in blood, suggesting that blood-based but brain cell-derived EVs can be a valuable source of biomarkers for neurodegenerative diseases. We have recently developed a new flow cytometry-based technology (Apogee) to analyze individual EVs carrying brain cell-specific and/or disease-related protein markers in body fluids for improved biomarker accuracy and utility. These sensitive and rapid assays directly quantify intact, individual EVs, without extensive purification before quantifying disease markers, which potentially reduce inter-lab variability and increasing the clinical utility of EV-based biomarkers. Further, in recent pilot studies, we and others observed that applying modified RT-QuIC assays to plasma EV samples could clearly distinguish between PD or MSA and healthy controls as well as between PD and MSA. In this study, we propose to further fine tune and optimize our Apogee and RT-QuIC assays to analyze EVs carrying both brain cell-specific markers (e.g., L1CAM or NMDAR2A for neurons, CNPase for oligodendrocytes, and GLAST for astrocytes) and disease-related markers (e.g., α-syn species, amyloid β and tau species). The optimized assays will then be validated for their performance in MSA and PD diagnosis and differential diagnosis in two independent plasma sample cohorts. These proposed experiments will likely establish two compensatory blood-based biomarker assays for MSA and PD – a rapid screening tool (Apogee) and a more accurate assay (RT-QuIC) to replace corresponding CSF assays. Both assays can be used in future larger-scale MSA and PD biomarker studies, which may establish the foundation leading to inexpensive and widely available blood tests to aid in PD/MSA diagnosis and/or disease tracking.
Dataset: Assessing the daily natural history of asymptomatic Plasmodium infections in adults and older children in Katakwi, Uganda: a longitudinal cohort study
(University of Washington, 2023-11-07) Hergott, Dianna; Egwang, Thomas; Owalla, Tonny; Murphy, Sean C; Staubus, W J; Seilie, A M; Chavtur, C; Chang, Ming; Balkus, J E; Apio, B; Lema, J; Cemeri, B; Akileng, A
This is the dataset accompanying Hergott et al. publications related to this field study conducted in Katakwi District, Uganda in March-May 2021. This dataset contains line listings of sample-by-sample data for Plasmodium RT-PCR results by subject.
Study Protocol: Feasibility of daily dried blood spot sampling to evaluate the natural history of low-density Plasmodium infections
(University of Washington, 2020-03-05) Murphy, Sean C; Hergott, Dianna; Egwang, Thomas; Owalla, Tonny
This document is the IRB-approved clinical trial protocol for a field study conducted in 2021 in Uganda. The findings of this study have been published (PMID: 35836179 and additional manuscripts in review).
High incidence of leukemia in large animals after stem cell gene therapy with a HOXB4-expressing retroviral vector
(2008-03-20) Zhang, Xiao-Bing; Beard, Brian C.; Trobridge, Grant D.; Wood, Brent L.; Sale, George E.; Sud, Reeteka; Humphries, Keith; Kiem, Hans-Peter
Retroviral vector–mediated HSC gene therapy has been used to treat individuals with a number of life-threatening diseases. However, some patients with SCID-X1 developed retroviral vector–mediated leukemia after treatment. The selective growth advantage of gene-modified cells in patients with SCID-X1 suggests that the transgene may have played a role in leukemogenesis. Here we report that 2 of 2 dogs and 1 of 2 macaques developed myeloid leukemia approximately 2 years after being transplanted with cells that overexpressed homeobox B4 (HOXB4) and cells transduced with a control gammaretroviral vector that did not express HOXB4. The leukemic cells had dysregulated expression of oncogenes, a block in myeloid differentiation, and overexpression of HOXB4. HOXB4 knockdown restored differentiation in leukemic cells, suggesting involvement of HOXB4. In contrast, leukemia did not arise from the cells carrying the control gammaretroviral vector. In addition, leukemia did not arise in 5 animals with high-level marking and polyclonal long-term repopulation following transplantation with cells transduced with an identical gammaretrovirus vector backbone expressing methylguanine methyltransferase. These findings, combined with the absence of leukemia in many other large animals transplanted with cells transduced with gammaretroviral vectors expressing genes other than HOXB4, show that HOXB4 overexpression poses a significant risk of leukemogenesis. Our data thus suggest the continued need for caution in genetic manipulation of repopulating cells, particularly when the transgene might impart an intrinsic growth advantage.
Toxicology evaluation of radiotracer doses of 3'-deoxy-3'-[18F]fluorothymidine (18F-FLT) for human PET imaging
(2007) Turcotte, Eric; Wiens, Linda W.; Grierson, John R.; Peterson, Lanell M.; Wener, Mark H.; Vesselle, Hubert
Background: 18F-FLT is a novel PET radiotracer which has demonstrated a strong potential utility for imaging cellular proliferation in human tumors in vivo. To facilitate future regulatory approval of 18F-FLT for clinical use, we wished to demonstrate the safety of radiotracer doses of 18F-FLT administered to human subjects, by: 1) performing an evaluation of the toxicity of 18F-FLT administered in radiotracer amounts for PET imaging, 2) comparing a radiotracer dose of FLT to clinical trial doses of FLT. Methods: Twenty patients gave consent to a 18F-FLT injection, subsequent PET imaging, and blood draws. For each patient, blood samples were collected at multiple times before and after 18F-FLT PET. These samples were assayed for a comprehensive metabolic panel, total bilirubin, complete blood and platelet counts. 18F-FLT doses of 2.59 MBq/Kg with a maximal dose of 185 MBq (5 mCi) were used. Blood time-activity curves were generated for each patient from dynamic PET data, providing a measure of the area under the FLT concentration curve for 12 hours (AUC12). Results: No side effects were reported. Only albumin, red blood cell count, hematocrit and hemoglobin showed a statistically significant decrease over time. These changes are attributed to IV hydration during PET imaging and to subsequent blood loss at surgery. The AUC12 values estimated from imaging data are not significantly different from those found from serial measures of FLT blood concentrations (p = 0.66). The blood samples-derived AUC12 values range from 0.232 ng*h/mL to 1.339 ng*h/mL with a mean of 0.802 � 0.303 ng*h/mL. This corresponds to 0.46% to 2.68% of the lowest and least toxic clinical trial AUC12 of 50 ng*h/mL reported by Flexner et al (1994). This single injection also corresponds to a nearly 3,000-fold lower cumulative dose than in Flexner's twice daily trial. Conclusion: This study shows no evidence of toxicity or complications attributable to 18F-FLT injected intravenously.
The urologic epithelial stem cell database (UESC) - a web tool for cell type-specific gene expression and immunohistochemistry images of the prostate and bladder
(2007) Pascal, Laura E.; Deutsch, Eric W.; Campbell, David S.; Korb, Martin; True, Lawrence D.; Liu, Alvin Y.
Background: Public databases are crucial for analysis of high-dimensional gene and protein expression data. The Urologic Epithelial Stem Cells (UESC) database http://scgap.systemsbiology.net/ is a public database that contains gene and protein information for the major cell types of the prostate, prostate cancer cell lines, and a cancer cell type isolated from a primary tumor. Similarly, such information is available for urinary bladder cell types. Description: Two major data types were archived in the database, protein abundance localization data from immunohistochemistry images, and transcript abundance data principally from DNA microarray analysis. Data results were organized in modules that were made to operate independently but built upon a core functionality. Gene array data and immunostaining images for human and mouse prostate and bladder were made available for interrogation. Data analysis capabilities include: (1) CD (cluster designation) cell surface protein data. For each cluster designation molecule, a data summary allows easy retrieval of images (at multiple magnifications). (2) Microarray data. Single gene or batch search can be initiated with Affymetrix Probeset ID, Gene Name, or Accession Number together with options of coalescing probesets and/or replicates. Conclusion: Databases are invaluable for biomedical research, and their utility depends on data quality and user friendliness. UESC provides for database queries and tools to examine cell typespecific gene expression (normal vs. cancer), whereas most other databases contain only whole tissue expression datasets. The UESC database provides a valuable tool in the analysis of differential gene expression in prostate cancer genes in cancer progression.
Differential expression of CD10 in prostate cancer and its clinical implication
(2007) Dall'Era, Marc A.; True, Lawrence D.; Siegel, Andrew F.; Porter, Michael P.; Sherertz, Tracy M.; Liu, Alvin Y.
Background: CD10 is a transmembrane metallo-endopeptidase that cleaves and inactivates a variety of peptide growth factors. Loss of CD10 expression is a common, early event in human prostate cancer; however, CD10 positive cancer cells frequently appear in lymph node metastasis. We hypothesize that prostate tumors expressing high levels of CD10 have a more aggressive biology with an early propensity towards lymph node metastasis. Methods: Eighty-seven patients, 53 with and 34 without pathologically organ confined prostate cancer at the time of radical prostatectomy (RP), were used for the study. Fourteen patients with lymph node metastasis found at the time of surgery were identified and included in this study. Serial sections from available frozen tumor specimens in OCT were processed for CD10 immunohistochemistry. Cancer glands were graded for the presence and intensity of CD10 staining, and overall percentage of glands staining positive was estimated. Clinical characteristics including pre- and post-operative PSA and Gleason score were obtained. A similar study as a control for the statistical analysis was performed with CD13 staining. For statistical analysis, strong staining was defined as > 20% positivity based on the observed maximum separation of the cumulative distributions. Results: CD10 expression significantly correlated with Gleason grade, tumor stage, and with preoperative serum PSA. Seventy percent of RP specimens from patients with node metastasis showed strong staining for CD10, compared to 30% in the entire cohort (OR = 3.4, 95% CI: 1.08-10.75, P = 0.019). Increased staining for CD10 was associated with PSA recurrence after RP. CD13 staining did not correlate significantly with any of these same clinical parameters. Conclusion: These results suggest that the expression of CD10 by prostate cancer corresponds to a more aggressive phenotype with a higher malignant potential, described histologically by the Gleason score. CD10 offers potential clinical utility for stratifying prostate cancer to predict biological behavior of the tumor.
Correlation of mRNA and protein levels: Cell type-specific gene expression of cluster designation antigens in the prostate
(2008) Pascal, Laura E.; True, Lawrence D.; Campbell, David S.; Deutsch, Eric W.; Risk, Michael; Coleman, Ilsa M.; Eichner, Lillian J.; Nelson, Peter S.; Liu, Alvin Y.
Background: Expression levels of mRNA and protein by cell types exhibit a range of correlations for different genes. In this study, we compared levels of mRNA abundance for several cluster designation (CD) genes determined by gene arrays using magnetic sorted and laser-capture microdissected human prostate cells with levels of expression of the respective CD proteins determined by immunohistochemical staining in the major cell types of the prostate - basal epithelial, luminal epithelial, stromal fibromuscular, and endothelial - and for prostate precursor/stem cells and prostate carcinoma cells. Immunohistochemical stains of prostate tissues from more than 50 patients were scored for informative CD antigen expression and compared with cell-type specific transcriptomes. Results: Concordance between gene and protein expression findings based on 'present' vs. 'absent' calls ranged from 46 to 68%. Correlation of expression levels was poor to moderate (Pearson correlations ranged from 0 to 0.63). Divergence between the two data types was most frequently seen for genes whose array signals exceeded background (> 50) but lacked immunoreactivity by immunostaining. This could be due to multiple factors, e.g. low levels of protein expression, technological sensitivities, sample processing, probe set definition or anatomical origin of tissue and actual biological differences between transcript and protein abundance. Conclusion: Agreement between these two very different methodologies has great implications for their respective use in both molecular studies and clinical trials employing molecular biomarkers.
Transcriptomes of human prostate cells
(2006) Oudes, Asa J.; Campbell, Dave S.; Sorensen, Carrie M.; Walashek, Laura S.; True, Lawrence D.; Liu, Alvin Y.
Background: The gene expression profiles of most human tissues have been studied by determining the transcriptome of whole tissue homogenates. Due to the solid composition of tissues it is difficult to study the transcriptomes of individual cell types that compose a tissue. To overcome the problem of heterogeneity we have developed a method to isolate individual cell types from whole tissue that are a source of RNA suitable for transcriptome profiling. Results: Using monoclonal antibodies specific for basal (integrin [Beta]4), luminal secretory (dipeptidyl peptidase IV), stromal fibromuscular (integrin [alpha] 1), and endothelial (PECAM-1) cells, respectively, we separated the cell types of the prostate with magnetic cell sorting (MACS). Gene expression of MACS-sorted cell populations was assessed with Affymetrix GeneChips. Analysis of the data provided insight into gene expression patterns at the level of individual cell populations in the prostate. Conclusion: In this study, we have determined the transcriptome profile of a solid tissue at the level of individual cell types. Our data will be useful for studying prostate development and cancer progression in the context of single cell populations within the organ.
Application of affymetrix array and massively parallel signature sequencing for identification of genes involved in prostate cancer progression
(2005) Oudes, Asa J.; Roach, Jared C.; Walashek, Laura S.; Eichner, Lillian J..; True, Lawrence D.; Vessella, Robert L.; Liu, Alvin Y.
Background: Affymetrix GeneChip Array and Massively Parallel Signature Sequencing (MPSS) are two high throughput methodologies used to profile transcriptomes. Each method has certain strengths and weaknesses; however, no comparison has been made between the data derived from Affymetrix arrays and MPSS. In this study, two lineage-related prostate cancer cell lines, LNCaP and C4-2, were used for transcriptome analysis with the aim of identifying genes associated with prostate cancer progression. Methods: Affymetrix GeneChip array and MPSS analyses were performed. Data was analyzed with GeneSpring 6.2 and in-house perl scripts. Expression array results were verified with RT-PCR. Results: Comparison of the data revealed that both technologies detected genes the other did not. In LNCaP, 3,180 genes were only detected by Affymetrix and 1,169 genes were only detected by MPSS. Similarly, in C4-2, 4,121 genes were only detected by Affymetrix and 1,014 genes were only detected by MPSS. Analysis of the combined transcriptomes identified 66 genes unique to LNCaP cells and 33 genes unique to C4-2 cells. Expression analysis of these genes in prostate cancer specimens showed CA1 to be highly expressed in bone metastasis but not expressed in primary tumor and EPHA7 to be expressed in normal prostate and primary tumor but not bone metastasis. Conclusion: Our data indicates that transcriptome profiling with a single methodology will not fully assess the expression of all genes in a cell line. A combination of transcription profiling technologies such as DNA array and MPSS provides a more robust means to assess the expression profile of an RNA sample. Finally, genes that were differentially expressed in cell lines were also differentially expressed in primary prostate cancer and its metastases.
Prostaglandin E2 receptor subtype 2 (EP2) regulates microglial activation and associated neurotoxicity induced by aggregated a-synuclein
(2007) Jin, Jinghua; Shie, Feng-Shiun; Liu, Jun; Wang, Yan; Davis, Jeanne; Schantz, Aimee M.; Montine, Kathleen S.; Moutine, Thomas J.; Zhang, Jing
Background: The pathogenesis of idiopathic Parkinson's disease (PD) remains elusive, although evidence has suggested that neuroinflammation characterized by activation of resident microglia in the brain may contribute significantly to neurodegeneration in PD. It has been demonstrated that aggregated a-synuclein potently activates microglia and causes neurotoxicity. However, the mechanisms by which aggregated a-synuclein activates microglia are not understood fully. Methods: We investigated the role of prostaglandin E2 receptor subtype 2 (EP2) in a-synuclein aggregation-induced microglial activation using ex vivo, in vivo and in vitro experimental systems. Results: Results demonstrated that ablation of EP2(EP2-/-) significantly enhanced microgliamediated ex vivo clearance of a-synuclein aggregates (from mesocortex of Lewy body disease patients) while significantly attenuating neurotoxicity and extent of a-synuclein aggregation in mice treated with a parkinsonian toxicant 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine. Furthermore, we report that reduced neurotoxicity by EP2-/- microglia could be attributed to suppressed translocation of a critical cytoplasmic subunit (p47-phox) of NADPH oxidase (PHOX) to the membranous compartment after exposure to aggregated a-synuclein. Conclusion: Thus, it appears that microglial EP2 plays a critical role in a-synuclein-mediated neurotoxicity.
Does the immune reaction cause malignant transformation by disrupting cell-to-cell or cell-to-matrix communications?
(2007) Prehn, Richmond T.
Tumor progression: In many (perhaps in all) tumor systems, a malignant cancer is preceded by a benign lesion. Most benign lesions do not transform to malignancy and many regress. The final transformative step to malignancy differs from the preceding steps in, among other things, that it often occurs in the absence of the original carcinogenic stimulus. Mechanism of immunostimulation: Relatively low titers of specific immune reactants are known to stimulate, but cell-to-cell or cell-to-matrix interactions appear to be major inhibitors of tumor-growth. Therefore, it seems reasonable to hypothesize that the mechanism of immunostimulation may be an interference with cell-to-cell or cell-to-matrix communication by a sub-lethal immune-reaction. Discussion: While the above hypothesis remains unproven, some evidence suggests that immunity may have a major facilitating effect on tumor growth especially at the time of malignant transformation. There is even some evidence suggesting that transformation in vivo may seldom occur in the absence of immunostimulation of the premalignant lesion. Positive selection by the immune reaction may be the reason that tumors are immunogenic.
Immunostimulation and Immunoinhibition of Premalignant Lesions
(2007) Prehn, Richmond T.
Background: The immune reaction may be either stimulatory or inhibitory to tumor growth, depending upon the local ratio of immune reactants to tumor cells. Hypothesis: A tumor-stimulatory immune response may be essential for survival of a neoplasm in vivo and for the biological progression from a premalignant lesion to a malignancy. Neither a positive nor a negative correlation between the magnitude of an immune-cell infiltrate and a cancer's prognosis can reveal whether the infiltrate was stimulating or inhibiting to the tumor's growth unless the position on the nonlinear curve that relates tumor growth to the magnitude of the immune reaction is known. Discussion: This hypothesis is discussed in relation to the development of human malignant melanomas and colorectal cancers.
The paradoxical effects of splenectomy on tumor growth
(2006) Prehn, Richmond T.
Background: There is a vast and contradictory literature concerning the effect of the spleen and particularly of splenectomy on tumor growth. Sometimes splenectomy seems to inhibit tumor growth, but in other cases it seems, paradoxically, to facilitate both oncogenesis and the growth of established tumors. Approach: In this essay I have selected from this large literature a few papers that seem particularly instructive, in the hope of extracting some understanding of the rules governing this paradoxical behavior. Conclusion: In general, whether splenectomy enhances or inhibits tumor growth seems to depend primarily upon the ratio of spleen to tumor. Small proportions of spleen cells usually stimulate tumor growth, in which case splenectomy is inhibitory. Larger proportions of the same cells, especially if they are from immunized animals, usually inhibit tumor growth, in which case splenectomy results in tumor stimulation.
An immune reaction may be necessary for cancer development
(2006) Prehn, Richmond T.
Background: The hypothesis of immunosurveillance suggests that new neoplasms arise very frequently, but most are destroyed almost at their inception by an immune response. Its correctness has been debated for many years. In its support, it has been shown that the incidences of many tumor types, though apparently not all, tend to be increased in immunodeficient animals or humans, but this observation does not end the debate. Alternative model: There is an alternative to the surveillance hypothesis; numerous studies have shown that the effect of an immune reaction on a tumor is biphasic. For each tumor, there is some quantitatively low level of immune reaction that, relative to no reaction, is facilitating, perhaps even necessary for the tumor's growth in vivo. The optimum level of this facilitating reaction may often be less than the level of immunity that the tumor might engender in a normal subject. Conclusion: The failure of a tumor to grow as well in the normal as it does in the immunosuppressed host is probably not caused by a lack of tumor-cell killing in the suppressed host. Instead, the higher level of immune response in a normal animal, even if it does not rise to tumor-inhibitory levels, probably gives less positive support to tumor growth. This seems more than a semantic distinction.
Prehn, R. On the nature of cancer and why anticancer vaccines don't work
(2005) Prehn, Richmond T.
In this essay I suggest that the major difficulty in producing effective anti-cancer vaccines lies in the fact that most cancers have little immunogenicity because of a basic paucity of tumor-specific antigenicity. The lack of antigenicity, despite extensive genomic instability, could be explained if most tumor mutations occur in silenced genes. A further problem is that an immune reaction against tumor antigens, especially in moderate or low amount, may be stimulatory rather than inhibitory to tumor growth.
The role of mutation in the new cancer paradigm
(2005) Prehn, Richmond T.
The almost universal belief that cancer is caused by mutation may gradually be giving way to the belief that cancer begins as a cellular adaptation that involves the local epigenetic silencing of various genes. In my own interpretation of the new epigenetic paradigm, the genes epigenetically suppressed are genes that normally serve in post-embryonic life to suppress and keep suppressed those other genes upon which embryonic development depends. Those other genes, if not silenced or suppressed in the post-embryonic animal, become, I suggest, the oncogenes that are the basis of neoplasia. Mutations that occur in silenced genes supposedly go unrepaired and are, therefore, postulated to accumulate, but such mutations probably play little or no causative role in neoplasia because they occur in already epigenetically silenced genes. These mutations probably often serve to make the silencing, and therefore the cancer, epigenetically irreversible.
Arbidol: a broad-spectrum antiviral that inhibits acute and chronic HCV infection
(2006) Boriskin, Yury S.; P�cheur, Eve-Isabelle; Polyak, Stephen J.
Arbidol (ARB) is an antiviral compound that was originally proven effective for treatment of influenza and several other respiratory viral infections. The broad spectrum of ARB anti-viral activity led us to evaluate its effect on hepatitis C virus (HCV) infection and replication in cell culture. Long-term ARB treatment of Huh7 cells chronically replicating a genomic length genotype 1b replicon resulted in sustained reduction of viral RNA and protein expression, and eventually cured HCV infected cells. Pre-treatment of human hepatoma Huh7.5.1 cells with 15 [micro]M ARB for 24 to 48 hours inhibited acute infection with JFH-1 virus by up to 1000-fold. The inhibitory effect of ARB on HCV was not due to generalized cytotoxicity, nor to augmentation of IFN antiviral signaling pathways, but involved impaired virus-mediated membrane fusion. ARB's affinity for membranes may inhibit several aspects of the HCV lifecycle that are membrane-dependent.
Identification of novel proteins affected by rotenone in mitochondria of dopaminergic cells
(2007) Jin, Jinghua; Davis, Jeanne; Zhu, David; Kashima, Daniel T.; Leroueil, Marc; Pan, Catherine; Montine, Kathleen S.; Zhang, Jing
Background: Many studies have shown that mitochondrial dysfunction, complex I inhibition in particular, is involved in the pathogenesis of Parkinson's disease (PD). Rotenone, a specific inhibitor of mitochondrial complex I, has been shown to produce neurodegeneration in rats as well as in many cellular models that closely resemble PD. However, the mechanisms through which complex I dysfunction might produce neurotoxicity are as yet unknown. A comprehensive analysis of the mitochondrial protein expression profile affected by rotenone can provide important insight into the role of mitochondrial dysfunction in PD. Results: Here, we present our findings using a recently developed proteomic technology called SILAC (stable isotope labeling by amino acids in cell culture) combined with polyacrylamide gel electrophoresis and liquid chromatography-tandem mass spectrometry to compare the mitochondrial protein profiles of MES cells (a dopaminergic cell line) exposed to rotenone versus control. We identified 1722 proteins, 950 of which are already designated as mitochondrial proteins based on database search. Among these 950 mitochondrial proteins, 110 displayed significant changes in relative abundance after rotenone treatment. Five of these selected proteins were further validated for their cellular location and/or treatment effect of rotenone. Among them, two were confirmed by confocal microscopy for mitochondrial localization and three were confirmed by Western blotting (WB) for their regulation by rotenone. Conclusion: Our findings represent the first report of these mitochondrial proteins affected by rotenone; further characterization of these proteins may shed more light on PD pathogenesis.
Cytogenetic characterization and H-ras associated transformation of immortalized human mammary epithelial cells
(2006) Rao, Krishna; Alper, Ozge; Opheim, Kent E.; Bonnet, George; Wolfe, Kristine; Larivee, Siobhan O'Hara; Porter, Peggy; McDougall, James K.
Introduction: Immortalization is a key step in malignant transformation, but immortalization alone is insufficient for transformation. Human mammary epithelial cell (HMEC) transformation is a complex process that requires additional genetic changes beyond immortalization and can be accomplished in vitro by accumulation of genetic changes and expression of H-ras. Methods: HMEC were immortalized by serial passaging and transduction with the catalytic subunit of the human telomerase gene (hTERT). The immortalized cells were passaged in vitro and studied by a combination of G- banding and Spectral Karyotyping (SKY). H-ras transduced, hTERT immortalized cells were cloned in soft agar and injected into nude mice. Extensive analysis was performed on the tumors that developed in nude mice, including immunohistochemistry and western blotting. Results: Immortal HMEC alone were not tumorigenic in [gamma]-irradiated nude mice and could not grow in soft agar. Late passage hTERT immortalized HMEC from a donor transduced with a retroviral vector containing the mutant, autoactive, human H-ras61L gene acquired anchorage independent growth properties and the capacity for tumorigenic growth in vivo. The tumors that developed in the nude mice were poorly differentiated epithelial carcinomas that continued to overexpress ras. These cells were resistant to doxorubicin mediated G1/S phase arrest but were sensitive to treatment with a farnesyltransferase inhibitor. Conclusion: Some of the cytogenetic changes are similar to what is observed in premalignant and malignant breast lesions. Despite these changes, late passage immortal HMEC are not tumorigenic and could only be transformed with overexpression of a mutant H-ras oncogene.

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