CombiFlow: Flow cytometry-based identification and characterization of genetically and functionally distinct AML subclones.

Many cancers, including leukemias, are dynamic oligoclonal diseases. Tools to identify and prospectively isolate genetically distinct clones for functional studies are needed. We describe our CombiFlow protocol, which is a combinatorial flow cytometry-based approach to identify and isolate such distinct clones. CombiFlow enables the visualization of clonal evolution during disease progression and the identification of potential relapse-inducing cells at minimal residual disease (MRD) time points. The protocol can be adapted to various research questions and allows functional studies on live sorted cell populations. For complete details on the use and execution of this protocol, please refer to de Boer et al. (2018).

Atividade anticarcinogênica da tributirina associada ou não ao sorafenibe em ratos Fischer-344 implantados com células GP7TB / Evaluation of anticarcinogenic activity associated or not with sorafenib in Fischer-344 rats implanted with GP7TB cells

O câncer primário de fígado (CPF) apresenta mau prognóstico, o que torna importante sua quimioprevenção. Nesse sentido, a tributirina (TB), um pró-fármaco do ácido butírico (AB), presente em laticínios e no mel, mostrou-se um agente quimiopreventivo promissor da hepatocarcinogênese experimental. Os efeitos inibitórios da TB têm sido relacionados à inibição do desenvolvimento de lesões pré-neoplásicas, bem como indução de apoptose e hiperacetilação de histonas. A quimioterapia é uma das abordagens mais comuns para o tratamento de diversos tipos de câncer, inclusive o CPF. Neste caso, o tratamento com sorafenibe (SO) é capaz de prolongar a sobrevida média dos pacientes com a doença em fases avançadas em aproximadamente apenas três meses. Em vista disso, são necessários estudos da associação do sorafenibe com outros compostos que possam aumentar a eficácia do tratamento quimioterápico. Desta forma, a associação de fármacos anti-neoplásicos com compostos bioativos dos alimentos pode consistir em uma estratégia potencial para aumentar a eficácia contra o câncer. No presente estudo, foi avaliada a atividade anticarcinogênica da TB e do SO, isoladamente ou em associação, na etapa de progressão da hepatocarcinogênese. Para tanto, foram realizados implantes singênicos no flanco de ratos Fischer-344 a partir de células da linhagem tumoral GP7TB. Quando as neoplasias atingiram 1 cm3, os animais foram aleatorizados em grupos experimentais: Grupo controle (CO), constituído por 10 ratos Fischer 344 que receberam Maltodextrina (300mg/ 100 g. p. c.), controle isocolarico e solução de etanol à 12,5% e Cremofor à 12,5% em agua estéril; Grupo Tributirina (TB), constituído por 9 ratos Fischer 344 que receberam TB (200mg/ 100 g. p. c.) e solução de etanol à 12,5% e Cremofor à 12,5% em água estéril; Grupo sorafenibe (SO) constituído por 9 ratos Fischer 344 que receberam Maltodextrina (300 mg/ 100 g. p. c.), controle isocalorico e tosilato de sorafenibe (3mg / 100 g. p. c. ) em água estéril; Grupo associação da tributirina com o sorafenibe (AS) constituído por 9 ratos Fischer 344 que receberam TB (20 mg/ 100 g. p. c.) e tosiliato de sorafenibe (3mg/ 100 g. p. c.); tratados por administração intragástrica (i.g) diariamente por 5 semanas consecutivas. As concentrações de AB e SO foram analisadas por cromatografia gasosa associada à espectrometria de massa e as neoplasias foram caracterizadas por imunoistoquímica. Em relação à evolução do tamanho das neoplasias o grupo AS apresentou menor (p=0,009) tamanho das mesmas em relação ao grupo CO. No entanto, estas diferenças não atingiram diferenças significativas (p>0,05) entre os grupos TB e CO, bem como entre os grupos SO e CO. Contudo, quando ajustados os valores do tamanho da neoplasia pela latência, observou-se alterações significativas (p<0,05) nos diversos grupos quando comparados ao grupo CO. O grupo SO aumentou a área necrótica das neoplasias, embora esta diferença não tenha atingido diferença significativa (p>0,05), enquanto que o grupo TB reduziu essa área necrótica em relação ao grupo CO (p=0,005). O grupo TB e AS apresentaram significativamente maiores (p<0,05) concentrações hepáticas e neoplásicas de AB em relação ao grupo CO. O grupo SO e AS apresentaram significativamente maiores (p<0,05) concentrações neoplásicas de SO em relação ao grupo CO. Os grupos SO e AS reduziram a expressão de PTEN, quando comparados ao grupo CO, embora esta diferença não tenha atingido diferença significativa (p>0,05). O grupo TB por sua vez expressou maiores niveis de PTEN, embora esta diferença não tenha atigindo significância estatística (p>0,05). Todos os grupos expressaram maiores niveis de caspase 3 clivada quando comparada ao grupo CO (p>0,05). OS grupos TB e SO reduziram a expressão de pERK ½ quando comparados ao grupo CO. embora estas diferenças não tenham atingidos diferença estatística (p>0,05). O grupo AS apresentou maior expressão de pERK ½ quando comparada ao grupo CO, embora esta diferença não tenha atingido diferença significativa (p>0,05). A caracterização das neoplasias do grupo CO foi padronizada por imunoistoquímica, apresentando-se positivas para CK 7, CK8, CK19 e Arginase e negativas para HepPar1 e CK18. Assim, os resultados sugerem que as neoplasias obtidas por implantes com células da linhagem GP7TB apresentam características de CPF oriundo de células tronco neoplásicas. Além disso, os grupos experimentais TB e AS apresentaram atividade anticarcinogênica promissora no modelo de implantes singênicos com células GP7TB, que eventualmente envolvem mecanismos de ação distintos da atividade quimioterápica apresentada pelo SO

Primary liver cancer (PLC) presents poor prognosis, which makes its chemoprevention important. In this sense, tributyrin (TB), a prodrug of butyric acid (AB), present in dairy products and honey, has been shown to be a promising chemopreventive agent for experimental hepatocarcinogenesis. The inhibitory effects of TB have been related to inhibition of the development of pre-neoplastic lesions, as well as induction of apoptosis and hyperacetylation of histones. Chemotherapy is one of the most common approaches for treating various types of cancer, including PLC. In this case, treatment with sorafenib (SO) is able to prolong the average survival of patients with the disease in advanced stages in approximately three months. In view of this, studies of the association of sorafenib with other compounds that may increase the efficacy of chemotherapeutic treatment are necessary. Thus, the association of anti-neoplastic drugs with bioactive compounds in food may be a potential strategy to increase efficacy against cancer. In the present study, the anticarcinogenic activity of TB and SO was evaluated, alone or in combination, in the progression stage of hepatocarcinogenesis. For this purpose, syngenic implants were performed on the flank of Fischer-344 mice from GP7TB tumor cells. When the neoplasms reached 1 cm3, the animals were randomized into experimental groups: Control group (CO), consisting of 10 Fischer 344 rats receiving Maltodextrin (300mg / 100 g.p.c), isocaloric control and 12.5% ethanol solution, and Cremofor to 12.5% in sterile water; Tributyrin group (TB), consisting of 9 Fischer 344 rats that received TB (200mg / 100 g.p.c.) and 12.5% ethanol solution and Cremofor 12.5% in sterile water; Sorafenib group (SO) consisting of 9 Fischer 344 rats receiving maltodextrin (300 mg / 100 g, w / w), isocaloric control and sorafenib tosylate (3 mg / 100 g, w / w) in sterile water; The association group of tributyrin and sorafenib (AS) consisted of 9 Fischer 344 rats receiving TB (20 mg / 100 g p.o.) and sorafenib tosylate (3 mg / 100 g p.o.); treated intragastric (i.g) daily for 5 consecutive weeks. The concentrations of AB and SO were analyzed by gas chromatography associated with mass spectrometry and the neoplasms were characterized by immunohistochemistry. In relation to the evolution of the size of the neoplasias, the AS group presented smaller (p = 0.009) size of the same ones in relation to the CO group. However, these differences did not reach significant differences (p> 0.05) between the TB and CO groups, as well as between the SO and CO groups. However, when adjusted for size of the neoplasm by latency, significant changes (p <0.05) were observed in the different groups when compared to the CO group. The SO group increased the necrotic area of the neoplasias, although this difference did not reach a significant difference (p> 0.05), while the TB group reduced this necrotic area in relation to the CO group (p = 0.005). The TB and AS groups presented significantly higher (p <0.05) hepatic and neoplastic AB concentrations than the CO group. The SO and AS groups presented significantly higher (p <0.05) neoplastic concentrations of SO in relation to the CO group. The SO and AS groups reduced the PTEN expression when compared to the CO group, although this difference did not reach a significant difference (p> 0.05). The TB group in turn expressed higher levels of PTEN, although this difference did not increase statistical significance (p> 0.05). All groups expressed higher levels of caspase 3 cleaved when compared to the CO group (p> 0.05). The TB and SO groups reduced the expression of pERK ½ when compared to the CO group. although these differences did not reach statistical difference (p> 0.05). The AS group presented higher pERK ½ expression when compared to the CO group, although this difference did not reach a significant difference (p> 0.05). Characterization of the neoplasias of the CO group was standardized by immunohistochemistry, presenting positive for CK 7, CK8, CK19 and Arginase and negative for HepPar1 and CK18. Thus, the results suggest that the neoplasias obtained by implants with GP7TB cells present CPF characteristics originating from neoplastic stem cells. In addition, the experimental groups TB and AS presented promising anticarcinogenic activity in the model of syngeneic implants with GP7TB cells, which eventually involve mechanisms of action distinct from the chemotherapy activity presented by SO

Systematic classification of melanoma cells by phenotype-specific gene expression mapping.

There is growing evidence that the metastatic spread of melanoma is driven not by a linear increase in tumorigenic aggressiveness, but rather by switching back and forth between two different phenotypes of metastatic potential. In vitro these phenotypes are respectively defined by the characteristics of strong proliferation/weak invasiveness and weak proliferation/strong invasiveness. Melanoma cell phenotype is tightly linked to gene expression. Taking advantage of this, we have developed a gene expression-based tool for predicting phenotype called Heuristic Online Phenotype Prediction. We demonstrate the predictive utility of this tool by comparing phenotype-specific signatures with measurements of characteristics of melanoma phenotype-specific biology in different melanoma cell lines and short-term cultures. We further show that 86% of 536 tested melanoma lines and short-term cultures are significantly associated with the phenotypes we describe. These findings reinforce the concept that a two-state system, as described by the phenotype switching model, underlies melanoma progression.

Quantitative assessment of hypoxia subtypes in microcirculatory supply units of malignant tumors using (immuno-)fluorescence techniques.

BACKGROUND AND PURPOSE: Hypoxia is a characteristic of tumors, is known to increase aggressiveness, and causes treatment resistance. Traditional classification suggests two types of hypoxia: chronic and acute. Acute hypoxia is mostly caused by transient disruptions in perfusion, while chronic hypoxia is caused by diffusion limitations. This classification may be insufficient in terms of pathogenetic and pathophysiological mechanisms. Therefore, we quantified hypoxia subtypes in tumors based on (immuno-)fluorescent marker distribution patterns in microcirculatory supply units (MCSUs). MATERIAL AND METHODS: Cryosections from hSCC lines (SAS, FaDu, UT-SCC-5, UT-SCC-14, UT-SCC-15) were analyzed. Hypoxia was identified by pimonidazole, perfusion by Hoechst 33342, and endothelial cells by CD31. The following patterns were identified in vital tumor tissue: (1) normoxia: Hoechst 33342 fluorescence around microvessels, no pimonidazole, (2) chronic hypoxia: Hoechst 33342 fluorescence around microvessels, pimonidazole distant from microvessels, (3) acute hypoxia: no Hoechst 33342 fluorescence around microvessels, pimonidazole in immediate vicinity of microvessels, and (4) hypoxemic hypoxia: Hoechst 33342 fluorescence and pimonidazole directly around microvessels. RESULTS: Quantitative assessment of MCSUs show predominance for normoxia in 4 out of 5 tumor lines (50.1-72.8%). Total hypoxia slightly prevails in UT-SCC-15 (56.9%). Chronic hypoxia is the dominant subtype (65.4-85.9% of total hypoxia). Acute hypoxia only accounts for 12.9-29.8% and hypoxemic hypoxia for 1.2-6.4% of total hypoxia. The fraction of perfused microvessels ranged from 82.5-96.6%. CONCLUSION: Chronic hypoxia is the prevailing subtype in MCSUs. Acute hypoxia and hypoxemic hypoxia account for only a small fraction. This approach enables assessment and recognition of different hypoxia subtypes including hypoxemic hypoxia and may facilitate methods to (clinically) identify and eliminate hypoxia.

[Cell phenotype determines PAI-1 antiproliferative effect - suppressed proliferation of the lung cancer but not prostate cancer cells]. / Fenotyp komórek warunkuje antyproliferacyjne dzialanie PAI-1 -hamujacy wplyw na aktywnosc proliferacyjna komórek raka pluca.

INTRODUCTION: Plasminogen inhibitor activator type 1 (PAI-1) is an important regulator of tumor growth and metastasis formation acting directly via specific urokinase complexing or indirectly due to its affinity to vitronectin. We have shown previously that PAI-1 modifies angiogenic activity of endothelial cells in a dose-dependent manner but also in close relationship to the cell phenotype. Present study aimed on evaluating the PAI-1 effect on the proliferative activity of lung cancer cells (A549), prostate cancer cells (DU145) as well as endothelial cells (HUVEC). RESULTS: Mutated PAI-1 (1, 10, 100 microg/mL) characterized by the prolonged antifibrinolytic activity (T1/2 approximately 7000 h) inhibited proliferation of lung cancer A549 cells in a dose-dependent (p < 0.001) and time-dependent (p < 0.001) manner. No significant effect on the DU145 prostate cancer cells has been observed except of the 72 h cultures with highest PAI-1 concentration (100 microg/ml) (p < 0.001). Proliferative activity of endothelial cells (HUVEC) was affected by 100 microg/ml PAI-1 only, and independent of the culture period (24, 48 and 72 h, p < 0.001). CONCLUSION: Plasminogen inhibitor activator type 1 modulates cell proliferation via antifibrynolitic mechanizm time- and dose-dependently, however final outcome is strongly affected by the cell phenotype.

Lineage relationship between LNCaP and LNCaP-derived prostate cancer cell lines.

BACKGROUND: LNCaP and its derivative cell lines, which include C4-2 (and the related C4-2B) and CL1, are used as models of prostate cancer. Unlike LNCaP, the other cell lines show features of progressed disease such as metastatic capability and hormone independence. Analyses were done to determine if C4-2 or CL1 cells were selected from pre-existent subpopulations in LNCaP. METHODS: Prostate cancer cells were characterized by cluster designation (CD) phenotyping. Specific cell populations were sorted by flow cytometry. DNA array analysis was used to probe differential gene expression. RESULTS: CD phenotyping showed that CL1 and C4-2 (and C4-2B) were very dissimilar, and C4-2 was more similar to LNCaP. One common difference between LNCaP and its derivatives was CD26, in which virtually all C4-2 or CL1 cells were CD26(+) but only approximately 10% of LNCaP cells were CD26(+). The CD26(+) subpopulation of LNCaP was isolated and cultured in vitro. After culture, a high percentage of the cells (descended from the sorted cells) were CD26(+), in contrast to those sorted by CD13 or CD44. The cultured CD13 and CD44 populations did not show a high percentage of CD13(+) and CD44(+) cells, respectively. CD13 and CD44 are markers, in addition to CD26, for CL1 but not for C4-2. CONCLUSIONS: C4-2 arose probably from CD26(+) LNCaP cells, while CL1 arose de novo.

Common origins of MDA-MB-435 cells from various sources with those shown to have melanoma properties.

Recently, the tissue origin of MDA-MB-435 cell line has been the subject of considerable debate. In this study, we set out to determine whether MDA-MB-435-DTP cells shown to express melanoma-specific genes were identical to various other MDA-MB-435 cell stocks worldwide. CGH-microarray, genetic polymorphism genotyping, microsatellite fingerprint analysis and/or chromosomal number confirmed that the MDA-MB-435 cells maintained at the Lombardi Comprehensive Cancer Center (MDA-MB-435-LCC) are almost identical to the MDA-MB-435-DTP cells, and showed a very similar profile to those obtained from the same original source (MD Anderson Cancer Center) but maintained independently (MDA-MB-435-PMCC). Gene expression profile analysis confirmed common expression of genes among different MDA-MB-435-LCC cell stocks, and identified some unique gene products in MDA-MB-435-PMCC cells. RT-PCR analysis confirmed the expression of the melanoma marker tyrosinase across multiple MDA-MB-435 cell stocks. Collectively, our results show that the MDA-MB-435 cells used widely have identical origins to those that exhibit a melanoma-like gene expression signature, but exhibit a small degree of genotypic and phenotypic drift.

Molecular biologic characteristics of seven new cell lines of squamous cell carcinomas of the head and neck and comparison to fresh tumor tissue.

BACKGROUND: Squamous cell carcinoma (SCC) is the most frequent malignant tumor of the upper aerodigestive tract. Cell lines of these tumors facilitate the investigation of various tumor biological parameters. This study was conducted to compare molecular biologic characteristics between cell lines and fresh tumor tissue. METHODS: In seven SCC-derived cell lines, cytokeratin 5/6 and cytokeratin 19 expression, DNA content, chromosome aberrations and tumorigenicity were assessed in nude rats. Unbalanced numerical and structural chromosomal aberrations were investigated by comparative genomic hybridization (CGH), and results were compared to those obtained in fresh tumor tissues of the same patients. RESULTS: All cell lines expressed cytokeratins 5/6 and 19, indicating their epidermoid origin. Tumor growth after transplantation into nude rats occurred in five of seven cell lines. Routine histology and immunohistochemical examinations confirmed SCC. Aneuploidy was detected in all cell lines, with a 2c deviation index ranging from 1.9 through 9.5 and a 5c exceeding rate ranging from 2.6 through 36.7%. The most frequent chromosomal aberrations in cell lines were overrepresentations of chromosomal material on chromosomes 15q, 7p (5 cases each), 3q, 5p (4 cases each), and 11q and 17q (3 cases each) and losses of chromosomal material on chromosomes 3p, 18q (3 cases each), and 19p and 7q (2 cases each). Comparing these results to CGH analysis of fresh tumor tissue from the same patients, overrepresentations of chromosomal material on 10q, 20q and 21q, along with loss of chromosomal material on 4q was detected more frequently in primary tumors, whereas overrepresentations on 7p and loss of chromosomal material on 7q were more frequently detected in cell lines. Nevertheless, there was a high degree of similarity of chromosomal alterations in cell lines and corresponding fresh tumor tissue. CONCLUSION: The data suggest a high degree of genetic similarity between tumor cells of cell lines and the tumors from which they were derived. Therefore, these cell lines can serve as an accurate model to investigate cell biology of SCC in vitro.

Large-scale sources of neural stem cells.

Large-scale sources of neural stem cells are crucial for both basic research and novel approaches toward treating neurological disorders. Three sources that produce neural cells closely resembling their normal counterparts are now available: oncogene immortalized stem cells, neurospheres, and embryonic stem cell (ES)-derived neural cells. Cells including multiple subtypes of CNS and PNS neurons, as well as oligodendrocytes, Schwann cells, and astrocytes, are modeled by these large-scale sources. Although most cell lines were originally from rodents, their human counterparts are being discovered and characterized.

Malignant gliomas display altered plasma membrane potential and pH regulation--interaction with Tc-99m-MIBI and Tc-99m-Tetrofosmin uptakes.

Two radiopharmaceuticals, Tc-99m-MIBI (MIBI) and Tc-99m-Tetrofosmin (Tfos), are currently used for in vivo non-invasive monitoring of the MultiDrug Resistant (MDR) status of tumours. As gliomas are highly multidrug resistant, it is expected that the tracers would be poorly retained in those cells, but the in vivo and in vitro studies to date have shown that Tfos was highly retained in malignant gliomas. The high degree of malignancy of tumour cells is linked to alterations of physiological parameters as plasma membrane potential and intracellular pH. In order to elucidate the contribution of those parameters to Tfos and MIBI uptakes in malignant gliomas, we used several glioma cell lines--G111, G5, G152, and 42 MG-BA. These cells showed to be chemoresistant with a high level of expression and activity of the Multidrug Resistant associated Protein 1 (MRP1). They also had an alkaline intracellular pH (pHi) related to the Na+/H+ antiporter (NHE-1) expression and depolarised plasma membranes (-45 to -55 mV). In spite of their chemoresistance, we have found a high accumulation of both radiotracers in gliomas, more important for Tfos than MIBI, related to the presence and activity of NHE-1. In conjunction, the uptakes of the tracers were only partially dependent upon the plasma membrane potential of the glioma cell lines, again Tfos uptake being less dependent on this parameter than MIBI uptake. In conclusion, the evidence accumulated in this study suggests that Tfos could be a suitable glioma marker in vivo.

Maturation sensitive and resistant t(15;17) NB4 cell lines as tools for APL physiopathology: nomenclature of cells and repertory of their known genetic alterations and phenotypes.

Chromosomal translocations, leading to gene rearrangements that generate chimerical proteins, represent one of the initiating events of leukemia. Preleukemia cells eventually develop into overt leukemia by occurrence of secondary genetic alterations (tumor progression). The physiopathology of leukemia has made considerable progress during the last two decades, due to molecular biology investigations on the role played by the altered genes, during neoplasic hemopoiesis. In vitro studies have been facilitated by the establishment of stable leukemia cell lines bearing these gene rearrangements and secondary gene mutations. Investigations on acute promyelocytic leukemia (APL) have benefited from maturation sensitive and resistant cell lines (NB4 and UF-1) derived from APL patient's leukemia cells and bearing the t(15;17). The information concerning the NB4 cell line (responsiveness to retinoid/rexinoid, cAMP, arsenic, mutations causing resistance) is spread in an abundant literature. In this paper, we briefly recapitulate the cellular and molecular features of this cell line and its subclones with the aim of facilitating investigators in their choice of the most appropriate tool for their studies. As redundancy of several names given to NB4 sublines has sometimes created difficulties, we propose a nomenclature for the various NB4 sublines that most investigators certainly would be agreed with.

The main biological determinants of tumor line taxonomy elucidated by a principal component analysis of microarray data.

By using principal components analysis (PCA) we demonstrate here that the information relevant to tumor line classification linked to the activity of 1375 genes expressed in 60 tumor cell lines can be reproduced by only five independent components. These components can be interpreted as cell motility and migration, cellular trafficking and endo/exocytosis, and epithelial character. PCA, at odds with cluster analysis methods routinely used in microarray analysis, allows for the participation of individual genes to multiple biochemical pathways, while assigning to each cell line a quantitative score reflecting fundamental biological functions.

Molecular identification of main cellular lineages as a tool for the classification of gastric cancer.

Gastric carcinomas (GC) are heterogeneous tumors comprising variable amounts of cells of different lineage phenotype, including gastric mucous cells (surface--SMC or gland--GMC) and intestinal cells (IC). The evaluation of tumor behavior has classically depended on strictly morphological classifications of tumors. Microsatellite instability (MSI) is frequently detected in GC, but whether MSI affects all gastric cellular lineages or exclusively occurs in unique cellular lineages in GC is not known. The aims of this study were to test a combination of anti-mucin antibodies to classify gastric cancer into predominant cell lineage phenotype and to determine whether MSI in GC is associated with particular cellular tumor phenotypes. Fifty-five GC were immunophenotyped with antibodies specific for SMC, GMC, or IC. DNA was extracted from tumor and non-neoplastic gastric tissues and amplified with 5 microsatellite markers. A mixed cellular pattern was the most frequent phenotype of GC (61%) and was seen in both glandular (63%) and diffuse (58%)-type tumors. No significant difference in the rate of MSI was found in tumors with predominant gastric, intestinal or mixed phenotype. However, tumors with null or low-level expression of cellular lineage differentiation markers displayed MSI more frequently than tumors with high-level expression (40% v 20%). In conclusion, different gastric carcinoma cell lineage patterns can be easily identified with the 3 immunohistochemical markers used in this study. The 3 main cellular lineage components of gastric cancer can be similarly affected by microsatellite instability, consistent with the notion that MSI is an early event in gastric carcinogenesis.

Cell density influences insulin-like growth factor I gene expression in a cell type-specific manner.

The effect of cellular density on insulin-like growth factor I (IGF-I) gene expression was characterized in several tumor-derived cell lines. IGF-I messenger RNA (mRNA) transcripts increased more than 200-fold when C6 glioma cells grew to postconfluence. IGF-I receptor and beta-actin mRNAs were induced by 6- and 2-fold, respectively, as a function of confluence. IGF-I mRNA transcripts in GH3 and SK-N-MC cells increased about 4- to 5-fold in confluent cultures compared with sparse cultures. In OVCAR-3 cells, the IGF-I mRNA level remained constant as the cell density increased. Transient transfection experiments were performed with IGF-I exon 1 promoter/luciferase fusion constructs in C6 cells. The luciferase activity of a construct containing exon 1 sequence between +75 and +282 (the most 5' transcription initiation site was designated +1) was stimulated by 2.5-fold in dense cultures compared with that in sparse cultures of C6 cells. Luciferase activities of other constructs containing at least 282 bp of exon 1 sequence were also stimulated about 2- to 4-fold by cell density. However, 3' deletion to +192 led to loss of the cell density stimulatory effect. In contrast, luciferase activities of IGF-I promoter constructs were not altered by cell density in SK-N-MC cells. When the conditioned medium of low density C6 cultures was exchanged with that of high density cultures, the IGF-I mRNA level remained the same. In summary, cell density has a cell type- and gene type-specific effect on IGF-I gene expression. A cell density response element(s) may be located between +192 and +282 of the exon 1 promoter region in C6 cells.

Systematic variation in gene expression patterns in human cancer cell lines.

We used cDNA microarrays to explore the variation in expression of approximately 8,000 unique genes among the 60 cell lines used in the National Cancer Institute's screen for anti-cancer drugs. Classification of the cell lines based solely on the observed patterns of gene expression revealed a correspondence to the ostensible origins of the tumours from which the cell lines were derived. The consistent relationship between the gene expression patterns and the tissue of origin allowed us to recognize outliers whose previous classification appeared incorrect. Specific features of the gene expression patterns appeared to be related to physiological properties of the cell lines, such as their doubling time in culture, drug metabolism or the interferon response. Comparison of gene expression patterns in the cell lines to those observed in normal breast tissue or in breast tumour specimens revealed features of the expression patterns in the tumours that had recognizable counterparts in specific cell lines, reflecting the tumour, stromal and inflammatory components of the tumour tissue. These results provided a novel molecular characterization of this important group of human cell lines and their relationships to tumours in vivo.

A gene expression database for the molecular pharmacology of cancer.

We used cDNA microarrays to assess gene expression profiles in 60 human cancer cell lines used in a drug discovery screen by the National Cancer Institute. Using these data, we linked bioinformatics and chemoinformatics by correlating gene expression and drug activity patterns in the NCI60 lines. Clustering the cell lines on the basis of gene expression yielded relationships very different from those obtained by clustering the cell lines on the basis of their response to drugs. Gene-drug relationships for the clinical agents 5-fluorouracil and L-asparaginase exemplify how variations in the transcript levels of particular genes relate to mechanisms of drug sensitivity and resistance. This is the first study to integrate large databases on gene expression and molecular pharmacology.

First searchable database for DNA profiles of human cell lines: sequential use of fingerprint techniques for authentication.

The authenticity and freedom from cross-contaminants of a cell line are important prerequisites for any research, development or production programs involving cell lines. Mini- and microsatellites in the human genome harboring variable-numbers of tandem repeat (VNTR) DNA markers allow individualization at the DNA level and are of practical value for genetic linkage mapping, forensic legal medicine, paternity testing, monitoring of bone marrow transplants, and individualization of established cell lines. We have validated fingerprint techniques of different single- and multiple-locus VNTRs enabling the establishment of a searchable database of DNA profiles. As a result, multiplexed polymerase chain reaction amplification fragment length polymorphism (AmpFLP) of four prominent and highly polymorphic minisatellite VNTR loci was proven as the best tool for screening the uniqueness of DNA profiles in a fingerprint database. In order to avoid false positivity, identical or similar DNA profiles based on AmpFLP VNTR were tested further using a multi-locus fingerprint system. Our data demonstrate that misidentification remains a chronic problem among human continuous cell lines (detailed information at URL http://www.dsmz.de). The combination of rapidly generated DNA profiles based on single-locus VNTR loci, their authentication by screening the fingerprint database, and confirmation of duplicate banding patterns using multilocus fingerprints constitute a highly reliable and robust method, which enables high fidelity and quality of maintenance independent from the quantity of individual cell lines.

Guidelines for the characterization and publication of human malignant hematopoietic cell lines.

Continuous human malignant hematopoietic (MH) cell lines have become invaluable tools for hematological diagnosis and research. Over the last 35 years several hundred cell lines spanning almost the whole spectrum of hematopoietic cell lineages have been described. The cardinal features of MH cell lines are their monoclonal origin, arrest of differentiation, genetic alterations, and unlimited proliferation; the major advantages of cell lines are the unlimited supply of cell material and the infinite storability and recoverability at will of the cells. Categorization of cell lines usually follows the physiological stages of hematopoietic differentiation in the various cell lineages. For an adequate classification, a detailed and comparative characterization of both primary and cultured cells is absolutely necessary. New cell lines, in particular, must be adequately characterized. While clinical and cell culture data and immunological and cytogenetic features are the most important data, cell lines should be described in as much additional detail as possible allowing any singular features to be pointed out. In addition to detailed characterization, immortality of the culture, proof of neoplasticity, authentication of the true origin of the cells, scientific significance and availability of the cell line for other investigators are of paramount importance. In summary, MH cell lines have the potential to greatly facilitate diverse studies of normal and malignant hematopoiesis; to that end, these cell lines must be extensively characterized and adequately described.

History and classification of human leukemia-lymphoma cell lines.

Continuous human leukemia-lymphoma cell lines have become indispensable tools in hematological research since the establishment of the first human lymphoma cell line Raji in 1963. We summarize here historical landmarks in the establishment of unique leukemia-lymphoma-derived cell lines from the various cell lineages; their special importance in hematopoietic research is emphasized. The first cell lines were derived from African Burkitt lymphomas and were found to integrate the Epstein-Barr virus in their genome leading to the discovery and isolation of this virus. However, it was later recognized that not every cell line derived from a patient with leukemia-lymphoma represents a malignant cell line as residual normal B-lymphocytes can also be immortalized by EBV infection. During the following 20-30 years many other types of hematopoietic cell lines, commonly derived from hematopoietic neoplasms, were established. These panels of cell lines now span almost the whole spectrum of hematopoietic cell lineages (except for dendritric cells) and the various distinct stages of differentiation along the respective cell axes. From early on, cell lines became important tools for basic and clinical hematological research, initially mainly in the field of immunology, but later expanding to other areas also. It became apparent that leukemia-lymphoma cell lines are of monoclonal origin, are arrested at a discrete maturational stage during differentiation in each lineage, and show sustained and growth factor-independent or -dependent unlimited proliferation. Categorization of cell lines might best be based on the physiological stages of hematopoietic differentiation in the various cell lineages. For an adequate classification, detailed characterizations of both the cell lines and the primary cells from which the cell lines originated are absolutely mandatory. In summary, the availability of large numbers of continuous leukemia-lymphoma cell lines has greatly facilitated clinical and immunobiological studies of normal and malignant hematopoiesis. Human leukemia-lymphoma cell lines will continue to provide exquisite model systems for many biomedical disciplines.