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B Hukku

Publications and source records attributed to B Hukku.

At least 19 recordsLinked to original sources
PubMed1998

Gene amplification and increased expression of the reduced folate carrier in transport elevated K562 cells.

The molecular bases for the 6-fold elevated methotrexate transport capacity of K562.4CF cells (Matherly et al., Cancer Res. 51: 3420-3426, 1991) were studied with reduced folate carrier (RFC) cDNA, genomic, and antibody probes. Southern analysis showed that RFC gene copies were increased (approximately 4- to 5-fold) in K562.4CF over wild-type K562 cells. Fluorescence in situ hybridization using a genomic RFC probe confirmed the localization of the RFC gene to the q-arm of chromosome 21. In K562.4CF cells, the frequent loss of a normal copy of chromosome 21 (61% of metaphases) was accompanied by RFC gene amplification and translocations of amplified RFC gene fragments to several (2 to 6) different chromosomal loci not seen in wild-type cells. Particularly intense RFC signals were mapped to homogeneously staining regions in chromosomes 2 and 15. Increased RFC gene copies were accompanied by a similar increase in the major 3.1 kb RFC transcript by northern blotting and an approximately 7-fold elevated level of the broadly migrating (80-95 kDa) RFC protein on a western blot probed with an RFC C-terminal peptide antibody. These results demonstrate that selection of cells with a growth-limiting concentration of reduced folates (0.4 nM of leucovorin) is sufficient to promote chromosomal aberrations, including gene amplification and translocations that result in increased RFC expression and folate transport.

Blotting, Western↗
PubMed1998

Cell line characterization and authentication.

Research and development involving the use of cell lines require precise knowledge of the purity and species of origin of the cell lines used. This can only be assured by periodic monitoring of cultured cell lines for possible contamination by other cells and for characteristics that authenticate the cell line identity. In the absence of such monitoring, inter- and intraspecies cell line contaminations are likely to occur in the laboratories of unsuspecting investigators and can result in the generation of mistaken conclusions with an attendant loss of investigators' time, effort, and resources. This chapter provides a history and an overview of the methods that have been developed for cell line authentication, the type of information each of these different methods provides, and how synthesis of that information can be used to characterize a cell line and confirm its identity. An effective cell line monitoring strategy is described that involves testing for a combination of genetic markers, including cell membrane species antigens, isoenzymes, chromosomes, and DNA fingerprints, and use of databases for each marker system to compare the results obtained with a test cell culture with results from an extensive panel of previously tested cell lines.

Animals↗
PubMed1998

Reduced folate carrier gene expression in childhood acute lymphoblastic leukemia: relationship to immunophenotype and ploidy.

Reduced folate carrier (RFC) transcripts in human leukemias were measured by a competitive PCR assay. Total RNAs were reverse transcribed and amplified in the presence of competitive templates for RFC and beta-actin. RFC transcripts were normalized to transcripts for beta-actin. In a series of K562 sublines, a approximately 30-fold range of RFC transcripts measured by PCR assay closely agreed with results of Northern analysis and varied in proportion to RFC protein on Western blots and [3H]methotrexate transport. RFC transcripts varied over a 88-fold range in 49 specimens from 48 children with acute lymphoblastic leukemia (ALL). Median RFC transcripts were similar for 15 T-cell and 33 B-precursor ALL samples (RFC/beta-actin = 6.13 x 10(-3) and 7.92 x 10(-3), respectively) and for 41 diagnostic (7.20 x 10(-3)) and 8 relapse (5.58 x 10(-3)) samples. Whereas PCR measurements of RFC transcripts approximated changes in methotrexate transport in B-precursor ALL blasts (n = 10), for T-ALL blasts (n = 12) there was no apparent relationship between these parameters. For hyperdiploid B-precursor blasts (n = 11) with greater than 52 chromosomes and three to five copies of chromosome 21, the median RFC transcript level was approximately 3-fold higher than that for diploid B-precursor blasts. RFC transcripts were also elevated for two of three B-precursor specimens with acquired trisomy 21. Our results suggest that RFC gene expression is far more predictive of methotrexate uptake capacity in B-precursor than T-ALL and that increased copies of chromosome 21 in B-precursor ALL blasts are generally associated with increased RFC transcripts. Hence, the good prognosis for children with hyperdiploid B-precursor ALL treated with antimetabolite-based chemotherapy and the high levels of methotrexate and methotrexate polyglutamates accumulated may, in part, reflect elevated RFC gene expression and capacities for methotrexate transport.

Adolescent↗
PubMed1997

Malignant transformation of human prostate epithelial cells by N-nitroso-N-methylurea.

We report the malignant transformation of adult human prostate epithelial cells after multiple exposures to the chemical carcinogen N-nitroso-N-methylurea. Such transformants showed morphological alterations and anchorage-independent growth in soft agar and induced carcinomas when transplanted into nude mice. No p53 or ras mutations were observed. Stepwise chromosomal changes in the progression to tumorigenicity were observed. Loss of the p arms of chromosome 8 (p10>pter) and chromosome 10 (p10>pter) and gain of the q arm of chromosome 8 (q10>qter) were only observed in the tumor outgrows. These findings provide the first evidence of malignant transformation of human prostate epithelial cells exposed to a chemical carcinogen.

Adult↗
PubMed1997

Chromosomal changes observed in immortalized human keratinocytes transformed by ionizing radiation.

Human epithelial cancer cells were induced by concerted action of DNA tumor virus and X-ray radiation. Treatment of nontumorigenic early passage AD12-SV40 immortalized epithelial cells (RHEK-1) at passage 23 with radiation, resulted in further changes in their growth properties. One day old cultures of these RHEK-1 cells were irradiated with graded doses of X-rays (0, 2, 4, 6, and 8 Gy i.e. RHEK-1, RHEK-1/200R, RHEK-1/400R, RHEK-1/600R, and RHEK-1/800R). Morphologic alterations, the ability to grow in soft agar, and to form rapidly-growing squamous cell carcinomas in nude mice were concomitantly acquired properties of the radiation transformed cell lines RHEK-1/200R and RHEK-1/ 400R. On the basis of commonality in having addition of some extra material in chromosome 11 in the region between q14/q22 in all tumorigenic cell lines RHEK-1/200R and RHEK-1/400R, and deletion of the same region in nontumorigenic irradiated cell lines-RHEK-1/600R and RHEK-1/800R, it is deduced this region may have some important oncogene/s or other gene/s that play an important role in tumorigenesis. When compared to squamous cell carcinoma data, the duplication observed in the present study is also observed in 28 to 38% of head and neck and also in 25% of cases of untreated malignant lesions of oral squamous cell carcinoma. Thus, this study shows the correlation between in vitro induced squamous cell carcinoma to in vivo tumors.

Animals↗
PubMed1997

Cytogenetic and molecular genetic analysis of tumorigenic human bronchial epithelial cells induced by radon alpha particles.

To establish a cell culture model for lung carcinogenesis, independent populations of the human papillomavirus 18-immortalized human bronchial epithelial cell line BEP2D were treated with high linear energy transfer radon-simulated alpha-particles, expanded and xenotransplanted into Nu/Nu mice. Six independent cell lines were established from tumors that developed from three separate radiation treatments as follows: treatment (Tx) 1 (30 cGy--two doses), H2BT, Tx 2 (30 cGy--single dose), R30T1L, R30T2 and R30T3L, Tx 3 (30 cGy--single dose), H1ATN and H1ATBA1. Cytogenetic analysis revealed common changes in all tumor lines: loss of the Y chromosome (ch), one of three copies of ch8, one of three copies of ch14, and one of two copies of ch4p16-pter and ch11p15-pter. Analysis of polymerase chain reaction-amplified short tandem repeats of informative loci confirmed the loss of chY in all lines and loss of heterozygosity (LOH) at eight loci spanning the length of ch8 in all lines from Tx's 1 and 2. Our data support previous studies indicating the presence of tumor suppressor genes on ch8. LOH also was confirmed on ch14 at locus D14S306 in all cell lines from Tx 2 and in one of two lines from Tx 3. This region, 14q12-q13, may contain changes in one of the five known somatostatin receptor genes (SSTR1). No LOH was detected at any of the informative loci tested for on ch4 or ch11.

Alpha Particles↗
PubMed1995

Alterations in p53 and p16INK4 expression and telomere length during spontaneous immortalization of Li-Fraumeni syndrome fibroblasts.

Normal cells have a strictly limited growth potential and senesce after a defined number of population doublings (PDs). In contrast, tumor cells often exhibit an apparently unlimited proliferative potential and are termed immortalized. Although spontaneous immortalization of normal human cells in vitro is an extremely rare event, we observed this in fibroblasts from an affected member of a Li-Fraumeni syndrome kindred. The fibroblasts were heterozygous for a p53 mutation and underwent senescence as expected at PD 40. In four separate senescent cultures (A to D), there were cells that eventually recommenced proliferation. This was associated with aneuploidy in all four cultures and either loss (cultures A, C, and D) or mutation (culture B) of the wild-type (wt) p53 allele. Loss of wt p53 function was insufficient for immortalization, since cultures A, B, and D subsequently entered crisis from which they did not escape. Culture C has continued proliferating beyond 400 PDs and thus appears to be immortalized. In contrast to the other cultures, the immortalized cells have no detectable p16INK4 protein. A culture that had a limited extension of proliferative potential exhibited a progressive decrease in telomere length with increasing PD. In the culture that subsequently became immortalized, the same trend occurred until PD 73, after which there was a significant increase in the amount of telomeric DNA, despite the absence of telomerase activity. Immortalization of these cells thus appears to be associated with loss of wt p53 and p16INK4 expression and a novel mechanism for the elongation of telomeres.

Animals↗
PubMed1993

Role of chromosome 5 in immortalization and tumorigenesis of human keratinocytes.

Rhim et al. were first to show that superinfection of Ad12-SV40-infected immortalized human epidermal cells with an RNA tumor virus containing a ras oncogene, such as Ki-MSV, or their treatment with chemical carcinogens, leads to the ability of cells to both grow in anchorage-independent fashion and to form tumors in athymic nude mice. We describe details of the chromosome changes observed during the transformation. The culture was monitored through 40 passages after Ad12-SV40 infection. Chromosomes 9 and 11 showed random monosomy during the initial stages, but by passage 10 clonal evolution of the cell line was well established. Observed chromosome monosomy/trisomy coupled with chromosome rearrangements (identified as chromosomes A through F) were monosomy 13, loss of p arms of 8 and 10, partial loss of 5 (del(5)(q13) and of the q arm of 18(del(18)(q12)), and extra copies of 11q, 20 and 21. During its progression to tumorigenicity, a derived chromosome E containing a segment of 5q, also appeared to play a major role. The cells remained immortalized as long as the 5q segment was present in some of the cell population as derived chromosomes E or F. Derivative chromosome E showed noteworthy changes during the progression to tumorigenicity, in both viral and chemical transformations. There was loss of heterozygosity of 5q due to an exchange of 5q with chromosomes E or F. In Ki-MSV- and 4NQO-transformed cells, presence of an altered chromosome E (identified as E1) was observed. In MNNG-treated cells, there was a selection of population of cells with further alteration in chromosome E (identified as E3). Besides alterations in chromosome E, additional chromosome changes leading to gene activation and amplification indicating a multistep progression to tumorigenicity were observed. The cytogenetic data reiterate the ever-increasing need for molecular analysis of nonrandom karyotype changes.

Carcinogens↗
PubMed1993

Radiation-induced deletion of chromosomal regions containing tumor suppressor genes in human bronchial epithelial cells.

While several specific genetic alterations have been associated with malignant transformation of human bronchial epithelial cells, they are not all present in every tumor and there is reason to believe that additional genes important for loss of replication control in these cells remain to be identified. In an effort to develop a human bronchial epithelial cell model suitable for identification and functional analysis of genes involved in loss of replication control, and for studying the genetic basis of the multi-stage phenotypic changes associated with tumorigenesis, we treated multiple independent populations of the human papillomavirus (HPV)-immortalized human bronchial epithelial cell line BEP2D with ionizing radiation. Following irradiation, cell lines representing the radiated populations were established from single soft agar-selected colonies. These lines--R2B5SA, R3B5DSA, R2H9S, R2H16S, R2H18S and R3D7S--were compared cytogenetically to the parent cell line and found to have new chromosomal deletions involving putative or confirmed tumor suppressor genes, including chromosome 13 in most R2B5SA cells and all R3B5SA cells, chromosomes 11p and 22 in R216S, and 3p, 11p and 22 in R2H18S. The R2B5SA cells that have lost one copy of chromosome 13 overgrow the culture but are not tumorigenic, suggesting that loss of a single copy of chromosome 13 confers growth advantage but not tumorigenicity. The data confirm that ionizing radiation induces many large chromosomal alterations including chromosomal loss, translocation and deletion and that following radiation it is possible to isolate immortalized nontumorigenic cell lines monosomic for regions known or suspected to contain tumor suppressor genes. The cell lines described here provide powerful models for further investigation of putative tumor suppressor genes including identification, functional analysis and stage of transformation at which they are operative.

Animals↗
PubMed1992

Human glucocorticoid receptor gene deletion following exposure to cancer chemotherapeutic drugs and chemical mutagens.

The sensitivity of the human glucocorticoid receptor (hGR) gene to mutagenesis by the cancer chemotherapeutic drugs adriamycin, bleomycin, and chlorambucil was evaluated using glucocorticoid-sensitive (dexs) subclones of the human leukemic cell line CEM-C7. Treatment of cells with either bleomycin or chlorambucil increased the frequency of glucocorticoid-resistant (dexr) clones 3.3- and 10-fold, respectively. Measurement of steroid-binding activity in intact dexr cells demonstrated that the predominant phenotype of drug-induced dexr clones was receptorless (r-). dexs CEM cells express only one functional hGR allele and, in addition, are heterozygous for a BclI restriction fragment length polymorphism in the hGR gene (L. A. Palmer and J. M. Harmon, Cancer Res., 51:5224-5231, 1991). To determine the basis of the r- phenotype, BclI digests of genomic DNA isolated from r+ and r- cell lines were examined for the presence of the polymorphic 2.4- and 4.4-kilobase digestion products. A deletion of all or part of the hGR gene was demonstrated by the absence of the 4.4-kilobase fragment in one of two bleomycin-induced dexr clones, as well as the ICR191-induced dexr cell line ICR27TK.3. Cytogenetic analysis of ICR27TK.3 showed that the distal portion of the long arm of one chromosome 5 had been replaced with a portion of chromosome 15. Thus, in at least two dexr cell lines, deletions and/or chromosome breaks in the hGR locus appear to account for the r- phenotype.

Alleles↗
PubMed1991

A human bronchial epithelial cell strain with unusual in vitro growth potential which undergoes neoplastic transformation after SV40 T antigen gene transfection.

Bronchial epithelial cells were cultured from an individual with no evidence of malignant disease. These cells, designated HB56B, had a greatly extended in vitro life-span, being able to undergo 50 passages and 200 population doublings in contrast to the usual 3 to 4 passages and 20 to 30 population doublings characteristic of normal human bronchial epithelial cells. HB56B cells had karyotypic evidence of an amplified region on the short arm of chromosome II. Unlike normal bronchial epithelial cells, which undergo terminal squamous differentiation in vitro in response to fetal bovine serum, HB56B cells were only minimally affected by serum. These cells were readily established as an immortalized cell line, HB56B/5T, following transfection with a plasmid containing SV40 early region DNA. HB56B cells were non-tumorigenic in athymic nude mice, but HB56B/5T cells within a few passages of transfection with the SV40 plasmid formed tumors of which 28/37 regressed. HB56B cells may offer an experimental system for the study of proliferation, differentiation, and senescence control in human bronchial epithelial cells.

Adult↗
PubMed1989

Comparative properties of untreated and N-nitrosobenzylmethyl-amine-transformed rat esophageal epithelial cell lines.

A culture system utilizing rat esophageal epithelial cells has been developed. Four normal and eight N-nitrosobenzylmethylamine-treated lines were compared with respect to chromosome number, anchorage-independent growth in agarose, and tumorigenic potential in syngeneic rats. All cell lines were aneuploid with nine in the near-tetraploid range and three in the near-diploid range. No relation between tumorigenic potential and chromosome number or structure was apparent. Similarly, anchorage-independent growth in agarose did not correlate with tumorigenic potential. Three of the 12 immortalized lines (two carcinogen-treated and 1 untreated) induced well-differentiated squamous cell carcinomas in syngeneic rats. These tumors had weak metastatic potentials suggesting that tumorigenic potential and metastatic ability are separately controlled. These cell lines will be useful for the investigation of factors involved in the conversion of immortalized rat esophageal epithelial cell lines to lines of high metastatic potential.

Animals↗
PubMed1989

A cell line derived from non-neoplastic human neuroretinal cells.

We have derived a cell line from an epiretinal membrane excised surgically from a premature female infant born at a gestational age of 25 weeks, and who developed stage 5 retinopathy of prematurity. The cell line, which in early passages appeared immunocytochemically to contain cells with both neuronal and glial characteristics, has been maintained in culture for 14 months at the time this manuscript was submitted, and has survived 20 passages. The cells have a diploid, human karyotype, with most cells possessing 46 normal appearing chromosomes, including 44 autosomes and two X-chromosomes. Morphologically, the cell line at early passages consisted of polygonal cells and also of cells possessing long, spindly branching processes. These two cell types were cloned. Nearly 100% of the cells of both morphologic types in mixed cultures stained immunocytochemically for neuron-specific enolase (NSE), a neuronal marker, and approximately 5-10% of the cells in mixed cultures (including about 50% of the cells with the spindly morphology, that were less prevalent in mixed cultures) stained for glial fibrillary acid protein (GFAP), a glial marker. We have not performed "double-label" immunocytochemistry, but it was evident from the proportion of cells that stained with each marker that many cells must contain both GFAP and NSE. At least 50% of the cells in most of the early cultures were positive for keratin, while all were (and remain) negative for muscle actin. No cells are found that are immunocytochemically positive for factor VIII, a vascular endothelial cell marker. These cultured cells have also been studied immunocytochemically for their production of extracellular matrix substances. The cultures are immunocytochemically positive for type IV (but not type I) collagen, laminin and fibronectin. In later passages, cells of both clones lost their immunocytochemical positivity for GFAP and NSE, and all became positive for keratin. Cells of both clones also developed a similar, polygonal morphology, lacking long processes. By electron microscopy, many of the cells were seen to possess nonmotile cilia, with a 9 + 0 pattern of microtubule doublets. This cell line may be useful for studies of human retinal cell development and metabolism, and responses to pathological processes.

Cell Division↗
PubMed1988

Nonrandom involvement of chromosome 4 in the progression of rat prostatic cancer.

Owing to progression of the original spontaneous Dunning R-3327 rat prostatic cancer, a large series of transplantable prostatic tumors have been isolated that differ widely in their histological degree of differentiation, growth rate, androgen sensitivity, and metastatic ability. Using these parameters as criteria, the full spectrum of disease progression is represented within this Dunning system of rat prostatic cancers, ranging from slow-growing, well-differentiated, androgen-sensitive, nonmetastatic forms to fast-growing, anaplastic, androgen-independent, highly metastatic forms. Cytogenetic analysis of the two least progressionally advanced Dunning cancers (i.e., histologically well-differentiated, slow-growing, nonmetastatic variants) demonstrated no structural or numerical chromosomal aberration, suggesting that the initial development of prostatic cancer may not require detectable cytogenetic changes. In contrast, all 16 of the progressionally more advanced Dunning variants analyzed had a series of characteristic structural and/or numerical chromosomal aberrations that minimally involved chromosome 4. This nonrandom involvement of chromosome 4 was consistently observed regardless of whether the karyotype of the cancer was near-diploid or hyperaneuploid, suggesting that chromosome 4 aberrations are specifically involved in the progression of rat prostatic cancer. In addition, all four variants that were highly metastatic had, besides aberration of chromosome 4, structural aberrations involving chromosomes 1, 2, and 11. Of the 14 variants that did not have a high metastatic ability, only two had a similar aberrations involving chromosomes 1, 2, 4, and 11, suggesting that these specific chromosomal aberrations may be necessary, albeit not sufficient, for a high metastatic ability of rat prostatic cancers.

Adenocarcinoma↗
PubMed1986

Gonadoblastoma presenting as isosexual precocious puberty in a genetic female.

A 6 1/2-year-old female with pseudoprecocious puberty was found to have a gonadoblastoma. Her karyotype was 46, XX; serum estradiol level was markedly elevated; and androgens were within the range of normal. The importance of the Y chromosome and dysgenetic gonad in the predisposition to development of a gonadoblastoma is reviewed. A further categorization of gonadoblastomas based on the presence or absence of the Y chromosome is suggested.

Child↗
PubMed1985

Development of a new human breast cancer cell line Ia-270.

A new human breast cancer cell line (Ia-270) has been isolated from a malignant pleural effusion from a woman with metastatic infiltrating ductal carcinoma of the breast. This cell line contains cytoplasmic estrogen (ER) and progesterone (PR) receptors. Following estradiol (E2) administration, PR synthesis is augmented and a higher level of saturation density is reached. In an athymic mouse, the cell line produced a tumor morphologically similar to the primary tumor. The results of isoenzyme and karyotype analyses demonstrate Ia-270 to be of human origin and free of HeLa cell contamination. The cell line has been maintained in continuous culture since April 1982 and may provide a useful in vitro system for studying the biology of human breast cancer.

Aged↗
PubMed1984

Cell characterization by use of multiple genetic markers.

The extensive use of cell cultures for diverse research purposes is one of the truly great international growth industries. With the proliferation of cells comes a responsibility for monitoring them for inter- and intraspecies characteristics. We use multiple genetic markers for cell identification, i.e. species specific antigens, isozymic phenotypes, chromosomal complement, and HL-A haplotypes. The methodologies employed are briefly described, and various examples cited to show how these markers can be utilized for cell line monitoring. Data are summarized from 275 cultures sent to our laboratory for analysis during the past eighteen months. The data show that, overall, 35% of the cultures received were contaminated. The majority of cell cultures submitted were human cell lines. We found that 36% of these cultures were cross contaminated; 25% by cells of another species and 11% by another human cell line. This high incidence of inter- and intraspecies contamination underscores the importance of frequent monitoring of cell cultures.

Animals↗