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L Hlatky

Publications and source records attributed to L Hlatky.

At least 19 recordsLinked to original sources
PubMed2005

Full-color painting reveals an excess of radiation-induced dicentrics involving homologous chromosomes.

PURPOSE: To determine the ratio of homologous to heterologous dicentric chromosomes induced in human cells by ionizing radiation. This ratio is influenced by, and thus potentially informative about, underlying DNA damage/repair/misrepair processes and also the geometry of individual chromosome domains within the interphase nucleus. MATERIALS AND METHODS: 24-color mFISH (multiplex fluorescent in situ hybridization) was used to determine the ratio of 1-color (homologous) to 2-color (heterologous) dicentrics produced in human lymphocytes or fibroblasts by gamma-rays, alpha particles, or iron ions at various doses. Assuming that randomness independent of homology holds, the expected homologue:heterologue ratio for diploid human male cells is approximately 0.024, as shown by deriving a formula applicable to simple interchanges and then extending the result, via Monte Carlo simulation, to the general situation where complex aberrations are also considered. RESULTS AND CONCLUSIONS: There was a substantial excess of homologous dicentrics, with probability of occurrence by chance less than 0.02 for each of the three radiations and only about 10(-8) for all the data combined. Overall, approximately 18 homologous dicentrics were expected but 47 were found, including 11 involving chromosome 1. Observed excesses were similar for both sparsely and densely ionizing radiations. Geometric proximity of homologues is a possible explanation for the overabundance; in that case more extensive statistics should eventually uncover a linear energy transfer (LET) dependence. An alternative possibility, not ruled out by the present data, is homology-dependent misrepair.

Cell Culture Techniques↗
PubMed2004

Chromosome spatial clustering inferred from radiogenic aberrations.

PURPOSE: Analysing chromosome aberrations induced by low linear energy transfer (LET) radiation in order to characterize systematic spatial clustering among the 22 human autosomes in human lymphocytes and to compare their relative participation in interchanges. MATERIALS AND METHODS: A multicolour fluorescence in situ hybridization (mFISH) data set, specifying colour junctions in metaphases of human peripheral blood lymphocytes 72 h after in vitro exposure to low LET radiation, was analysed separately and in combination with previously published results. Monte Carlo computer simulations and mathematical modelling guided data analysis. RESULTS AND CONCLUSIONS: Statistical tests on aberration data confirmed two clusters of chromosomes, [1, 16, 17, 19, 22] and [13, 14, 15, 21, 22], as having their members being on average closer to each other than randomness would predict. The first set has been reported previously to be near the centre of the interphase nucleus and to be formed mainly by gene-rich chromosomes, while the second set comprises the nucleolus chromosomes. The results suggest a possible interplay between chromosome positioning and transcription. A number of other clusters suggested in the literature were not confirmed and considerable randomness of chromosome-chromosome juxtapositions was present. In addition, and consistent with previous results, it was found that chromosome participation in interchanges is approximately proportional to the two-thirds power of the DNA content.

Chromosome Aberrations↗
PubMed2004

Quantitative analysis of radiation-induced chromosome aberrations.

We review chromosome aberration modeling and its applications, especially to biodosimetry and to characterizing chromosome geometry. Standard results on aberration formation pathways, randomness, dose-response, proximity effects, transmissibility, kinetics, and relations to other radiobiological endpoints are summarized. We also outline recent work on graph-theoretical descriptions of aberrations, Monte-Carlo computer simulations of aberration spectra, software for quantifying aberration complexity, and systematic links of apparently incomplete with complete or truly incomplete aberrations.

Animals↗
PubMed2002

Computer analysis of mFISH chromosome aberration data uncovers an excess of very complicated metaphases.

PURPOSE: To analyse spectra of chromosome aberrations induced in vitro by low LET radiation, in order to characterize radiation damage mechanisms quantitatively. METHODS: Multiplex fluorescence in situ hybridization (mFISH) allows the simultaneous identification of each homologous chromosome pair by its own colour. mFISH data, specifying number distributions for colour junctions in metaphases of human peripheral blood lymphocytes 72 hours after exposure in vitro to a 3 Gy gamma-ray dose, were combined with similar, previously published results. Monte Carlo computer implementations of radiobiological models for chromosome aberration production guided quantitative analyses, which took into account distribution of cells among different metaphases and lethal effects or preferential elimination of some aberrations at cell division. RESULTS AND CONCLUSIONS: Standard models of DNA damage induction/repair/misrepair explain the main trends of the data as regards the fraction of metaphases having a particular number of colours involved in colour junctions. However, all standard models systematically under-predict the observed fraction of metaphases where a large number of different chromosomes participate in aberrations. An early appearance of chromosomal instability could explain most of the discrepancies.

Chromosome Aberrations↗
PubMed2001

Large-mutation spectra induced at hemizygous loci by low-LET radiation: evidence for intrachromosomal proximity effects.

A mathematical model is used to analyze mutant spectra for large mutations induced by low-LET radiation. The model equations are based mainly on two-break misrejoining that leads to deletions or translocations. It is assumed, as a working hypothesis, that the initial damage induced by low-LET radiation is located randomly in the genome. Specifically, we analyzed data for two hemizygous loci: CD59- mutants, mainly very large-scale deletions (>3 Mbp), in human-hamster hybrid cells, and data from the literature on those HPRT- mutants which involve at least deletion of the whole gene, and often of additional flanking markers (approximately 50-kbp to approximately 4.4-Mbp deletions). For five data sets, we estimated f, the probability that two given breaks on the same chromosome will misrejoin to make a deletion, as a function of the separation between the breaks. We found that f is larger for nearby breaks than for breaks that are more widely separated; i.e., there is a "proximity effect". For acute irradiation, the values of f determined from the data are consistent with the corresponding break misrejoining parameters found previously in quantitative modeling of chromosome aberrations. The value of f was somewhat smaller for protracted irradiation than for acute irradiation at a given total dose; i.e., the mutation data show a decrease that was smaller than expected for dose protraction by fractionation or low dose rate.

Animals↗
PubMed2000

Her-2-neu expression and progression toward androgen independence in human prostate cancer.

BACKGROUND: Human prostate cancers are initially androgen dependent but ultimately become androgen independent. Overexpression of the Her-2-neu receptor tyrosine kinase has been associated with the progression to androgen independence in prostate cancer cells. We examined the expression of Her-2-neu in normal and cancerous prostate tissues to assess its role in the progression to androgen independence. METHODS: Prostate cancer tissue sections were obtained from 67 patients treated by surgery alone (UNT tumors), 34 patients treated with total androgen ablation therapy before surgery (TAA tumors), and 18 patients in whom total androgen ablation therapy failed and who developed bone metastases (androgen-independent [AI] disease). The sections were immunostained for Her-2-neu, androgen receptor (AR), prostate-specific antigen (PSA), and Ki-67 (a marker of cell proliferation) protein expression. Messenger RNA (mRNA) levels and gene amplification of Her-2-neu were examined by RNA in situ hybridization and fluorescent in situ hybridization(FISH), respectively, in a subset of 27 tumors (nine UNT, 11 TAA, and seven AI). All statistical tests were two-sided. RESULTS: Her-2-neu protein expression was statistically significantly higher in TAA tumors than in UNT tumors with the use of two different scoring methods (P =.008 and P =.002). The proportion of Her-2-neu-positive tumors increased from the UNT group (17 of 67) to the TAA group (20 of 34) to the AI group (14 of 18) (P<.001). When compared with UNT tumors, tumor cell proliferation was higher in AI tumors (P =.014) and lower in TAA tumors (P<.001). All tumors expressed AR and PSA proteins. Although Her-2-neu mRNA expression was high in TAA and AI tumors, no Her-2-neu gene amplification was detected by FISH in any of the tumor types. CONCLUSIONS: Her-2-neu expression appears to increase with progression to androgen independence. Thus, therapeutic targeting of this tyrosine kinase in prostate cancer may be warranted.

Androgens↗
PubMed1999

Tumor development under angiogenic signaling: a dynamical theory of tumor growth, treatment response, and postvascular dormancy.

The effects of the angiogenic inhibitors endostatin, angiostatin, and TNP-470 on tumor growth dynamics are experimentally and theoretically investigated. On the basis of the data, we pose a quantitative theory for tumor growth under angiogenic stimulator/inhibitor control that is both explanatory and clinically implementable. Our analysis offers a ranking of the relative effectiveness of these inhibitors. Additionally, it reveals the existence of an ultimate limitation to tumor size under angiogenic control, where opposing angiogenic stimuli come into dynamic balance, which can be modulated by antiangiogenic therapy. The competitive influences of angiogenically driven growth and inhibition underlying this framework may have ramifications for tissue size regulation in general.

Angiogenesis Inhibitors↗
PubMed1999

Distinct mathematical behavior of apoptotic versus non-apoptotic tumor cell death.

PURPOSE: The presence or absence of a p53-dependent apoptosis response has previously been shown to greatly influence radiosensitivity in tumor cells. Here, we examine clonogenic survival curves for two genetically related oncogene transformed cell lines differing in the presence or absence of p53 and apoptosis. Solid tumor radiosensitivity patterns have been previously described for these lines. MATERIALS AND METHODS: Oncogene-transformed fibroblasts derived from E1A + Ras transfection of p53-wild-type or p53-null mouse embryonic fibroblasts were plated as single cells and irradiated at increasing radiation doses in single fractions from 1.5 to 11 Gy. Clonogenic cell survival assays were obtained. Survival data are fit to a linear-quadratic relationship: S = e(-alphaD-betaD2). Apoptosis was assessed and quantitated morphologically by staining with the fluorescent nuclear dye DAPI, by TUNEL assay for DNA fragmentation, and by measurement of apoptotic cysteine protease cleavage activity in cytosolic extracts. RESULTS: Whereas radiation triggers massive apoptosis in the presence of p53, it produces no measurable DNA fragmentation, apoptotic cysteine protease cleavage activity, or morphological changes of apoptosis in the cells lacking p53. These contrasting mechanisms of death display dramatically different quantitative behavior: log-survival of apoptotic cells is linearly proportional to dose (S = e(-alphaD)), whereas survival of non-apoptotic (p53 null) is linear-quadratic with a significant quadratic contribution. The surviving fraction at 2 Gy (SF-2) for p53-null cells was 70% verses 12% for p53-intact cells. CONCLUSIONS: In this system, apoptosis appears to exhibit a dominance of single-event which produces a very high alpha/beta ratio, and no significant shoulder; whereas non-apoptotic death in this system exhibits a comparatively small linear component, a low alpha/beta ratio, and a larger shoulder.

Animals↗
PubMed1998

Adeno-associated virus-mediated delivery of antiangiogenic factors as an antitumor strategy.

Antiangiogenic tumor therapies have recently attracted intense interest for their broad-spectrum action, low toxicity, and, in the case of direct endothelial targeting, an absence of drug resistance. To promote tumor regression and to maintain dormancy, antiangiogenic agents need to be chronically administered. Gene therapy offers a potential way to achieve sustained therapeutic release of potent antiangiogenic substances. As a step toward this goal, we have generated recombinant adeno-associated virus (rAAV) vectors that carry genes coding for angiostatin, endostatin, and an antisense mRNA species against vascular endothelial growth factor (VEGF). These rAAVs efficiently transduced three human tumor cell lines tested. Transduction with an rAAV-encoding antisense VEGF mRNA inhibited the production of endogenous tumor cell VEGF. Conditioned media from cells transduced with this rAAV or with rAAV-expressing endostatin or angiostatin inhibited capillary endothelial cell proliferation in vitro. Antiangiogenic rAAVs may offer a novel gene therapy approach to undermining tumor neovascularization and cancer progression.

Angiostatins↗
PubMed1998

Cell resensitization during protracted dosing of heterogeneous cell populations.

When a tumor or other heterogeneous cell population is acutely exposed to ionizing radiation (or, for that matter, to chemotherapeutic agents or hyperthermia), cells that happen to be more sensitive will be preferentially removed, leaving behind a population more resistant as a whole. However, under broadly applicable assumptions, we here demonstrate mathematically that there is a natural tendency of the postirradiation population to recover from the irradiation in such a manner as to restore its original sensitivity composition, i.e. to undergo "resensitization". An important consequence in radiotherapy is that, if a fixed total radiation dose is delivered in a more protracted manner, e.g. as several fractions or as a continuous dose at low dose rate, resensitization occurring over the course of dose delivery will result in greater cell killing than would otherwise have occurred. That is, for a cell population with any form of diversity in radiosensitivity, the influence of redistribution is to make any prolonged dose more damaging than an acute dose of the same magnitude. This tendency toward an "inverse dose-rate effect" may be masked in practice by countervailing effects, such as repair of sublethal damage, but the tendency is demonstrated to hold under very general circumstances, being a consequence of cell-cell diversity and the dynamic response of the cell population to treatment.

Animals↗
PubMed1997

Heterogeneity of androgen receptor content in advanced prostate cancer.

The principal mode of treatment of advanced (late stage) prostate cancer is androgen ablation. Although the response rate to hormonal ablation is high, relapse ultimately leading to death occurs in the majority of patients in remission from outgrowth of androgen-independent tumor cells. High-grade and high-stage cancers are more likely to progress to androgen independence. This study was undertaken to analyze the expression level of androgen receptor (AR) protein in prostatic carcinomas in relationship to grade and stage of disease. AR protein expression was assessed in 40 archival cases of prostate carcinoma by automated immunohistochemical techniques with standardized development times. Positive nuclei were quantitated by computer-assisted image analysis. Eighty-five percent of the prostatic carcinomas showed high levels of expression, defined as having AR present in more than 50% of the cells by light microscopy. Results of image analysis demonstrated that the variability of AR protein content per unit nuclear area increased with increasing grade (P < .03), regardless of cell size. High-grade prostatic intraepithelial neoplasia (PIN), present in 17 (42.5%) of the 40 cases, showed markedly reduced AR nuclear staining, compared with low-grade PIN or normal prostate. We show that AR content in prostate tumor cells becomes more variable with increasing Gleason score. In high-grade PIN, the in situ precursor of invasive prostate cancer, AR expression is either downregulated and/or restricted to the cytoplasm, but it is not heterogeneous. These data suggest that the heterogeneity in the expression of the receptor increases with progression of invasive prostate cancer and might in part account for a variable response to endocrine therapy.

Adult↗
PubMed1996

Measurement of potential doubling time for human tumor xenografts using the cytokinesis-block method.

Estimates of the potential doubling time (Tpot) of seven human tumor xenografts were made using a cytokinesis-block method. This method is currently being investigated as an alternative to flow cytometric assays using the administration of a thymidine analog in the measurement of Tpot. If perfected, the cytokinesis-block method of measuring Tpot would be advantageous as a predictive assay, in that no label is administered to the tumors in situ. Xenografts were grown in nude mice, and following tumor excision and disaggregation, tumor cells were cultured with the cytokinesis-blocking agent cytochalasin B. The flux of cells through mitosis was marked by the accumulation of multinucleate cells. By counting the total number of nuclei as a function of time, the effective population growth was observed. Tpot values were obtained by fitting suitable exponential least squares curves to the data, with the doubling time indicated by the fitted functions. For the seven tumors studied, a significant spread in growth rates was observed. Tpot values generated by this method ranged from approximately 2 days for a rapidly growing squamous cell carcinoma of the pharynx, FaDu, to approximately 7.5 days for the slower growing glioblastoma multiforme U251-MG. These values are compared with standard 5-iododeoxyuridine Tpot measures and volume doubling times obtained by Perez et al. (Cancer Res., 55: 392-398, 1995) for the same tumor xenografts. Although individual Tpot values varied between these methods, the ranking of the seven tumors in order of Tpot times was the same regardless of method. In addition to estimating Tpot, for each of the tumors, the fraction of clonogenically dead cells that was microscopically apparent, including apoptotic cells and cells expressing micronuclei, was determined as a function of time in culture. Tracking this in vitro cell loss rate provides information on the adjustment of these primary tumor cells to in vitro culture, a factor that needs to be addressed when determining how in vitro measurements of Tpot can be effectively related to in vivo measurements.

Animals↗
PubMed1996

Resensitization due to redistribution of cells in the phases of the cell cycle during arbitrary radiation protocols.

When a cell population in exponential growth is subjected to ionizing radiation, the degree to which its long-term size is attenuated, relative to a control population that is not irradiated, depends not only on the total dose but also on the time pattern of dose delivery. Using a standard mathematical model for cycling cell populations with age-dependent radiosensitivity, it has recently been shown that normal progression of cells through the cycle tends to decrease this relative population size when the total dose delivery time is increased from essentially zero times to short, finite times (Chen et al., Math. Biosci. 126, 147-170, 1995). This mathematical result is an agreement with intuitive arguments and experiments long known in radiobiology. Mechanistically, it says that after the first part of a dose has preferentially eliminated the more sensitive cells of an exponentially cycling cell population, cell cycle progression, with the consequent redistribution of cells among cycle phases, tends to "resensitize" that population, an affect countering that of sublethal damage repair. The present paper now generalizes this result, demonstrating that the redistribution-induced increase of cell killing carries over to doses of arbitrary duration. That is to say, delivering a given dose over some extended period will result in lesser ultimate population size (i.e. population size measured at some fixed time long after irradiation has ceased) than will delivering the same total dose acutely. The redistribution-induced resensitization occurs no matter how radiosensitivity depends on cell age. For illustration, examples are given to show that, for a split dose, the least sensitivity is observed when the two doses coincide. These examples also demonstrate, within the constraints of the overall resensitization principle, the possibility of an oscillatory dependence of population sensitivity on interfraction time.

Cell Cycle↗
PubMed1995

Quantifying intracellular radioresponse diversity in irradiated sandwich cultures via micronucleus expression.

Determining the degree of diversity in therapeutic sensitivity exhibited by a tumour population is of considerable clinical importance. In addition to being a contributor to radiation resistance, diversity is the basis for variation in sensitivity over the course of treatment. To study intrapopulation diversity in radiosensitivity following gamma-irradiation (2 Gy), distributions of the number of micronuclei/binucleate cell were obtained for human cervix carcinoma sandwich populations. Cell-to-cell diversity in radioresponse (micronucleus expression) was quantified using the overdispersion index ((variance/mean)--1). As measured by this index, the radioresponse diversity of sandwich cultures sharply increased after introduction of oxygen/nutrients to the cultures, mimicking tumour reperfusion. In addition, a strong correlation was found between this measure of diversity and the extent to which the fraction of cells without micronuclei exceeds that expected from a Poisson distribution. This correlation indicates that for a diverse population there can be a significant departure of the aggregate population sensitivity (determined, for instance, by log-survival in a clonogenic assay) from that inferable from simply averaging per-cell sensitivities (reflected, e.g. by mean number of chromosome aberrations/cell). Our experimental results suggest a model attributing diversity in a population to its being a mixture of distinct subpopulations, each biologically homogeneous with respect to micronucleus expression, and each contributing an individual Poisson-distributed micronucleus response. We demonstrate how such radiodiversity may be quantified and show that reoxygenation of a microenvironmentally heterogeneous population leads to an increase in its radiobiological diversity.

Cell Cycle↗
PubMed1994

Mammary fibroblasts may influence breast tumor angiogenesis via hypoxia-induced vascular endothelial growth factor up-regulation and protein expression.

Recent studies demonstrate the relationship of microvessel density to malignant progression in breast cancer (N. Weidner, J. P. Semple, W. R. Welch, and J. Folkman, N. Engl. J. Med., 324: 1-8, 1991), underscoring the importance of angiogenesis in this tumor. Crucial in tumor angiogenesis are the paracrine actions of tumor-secreted factors (e.g., vascular endothelial growth factor), which have been thought to derive from the tumor epithelial cells themselves. We demonstrate that in response to hypoxic conditions, human mammary fibroblasts dramatically up-regulate vascular endothelial growth factor mRNA and increase vascular endothelial growth factor protein levels in accordance with the degree of oxygen deprivation. Thus, mammary stromal cells, only recently considered in the regulation of breast carcinomas, may play a hitherto unrealized role in breast cancer angiogenesis.

Breast↗