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Biomedical subjects

P Hahnfeldt

Publications and source records attributed to P Hahnfeldt.

At least 19 recordsLinked to original sources
PubMed2000

Underprediction of visibly complex chromosome aberrations by a recombinational-repair ('one-hit') model.

PURPOSE: Published low-LET FISH data were used to test two models of chromosome aberration production based on breakage-and-reunion or recombinational repair. MATERIALS AND METHODS: Randomness of DNA double strand break induction and misrejoining is analyzed comprehensively and adopted as a working hypothesis. Proximity effects are approximated by using interaction sites. Model results are calculated using CAS (chromosome aberration simulator) Monte Carlo computer software with two adjustable parameters. CAS can emulate the specifics of any experimental painting protocol, allowing very detailed tests of the models. RESULTS: To reasonable approximation, breakage-and-reunion model predictions are consistent with low-LET FISH results, including two large, elaborate, one-paint data sets. An explicitly specified version of the recombinational-repair model severely underpredicts the frequency of the visibly complex aberration patterns most commonly observed with one-paint FISH, and is inconsistent with some observed multi-paint patterns. When high-dose effects (distortion and saturation) are taken into account quantitatively, a dose-response relation for apparently simple interchanges slightly favours the breakage-and-reunion model over the recombinational-repair model, despite being approximately linear over the dose range 2-6 Gy. CONCLUSIONS: The random breakage-and-reunion model gives comprehensive baseline predictions that are sufficiently accurate for the organization of experimental results. The data speak against complex aberrations being formed by the random recombinational repair pathway discussed here.

Chromosome Aberrations↗
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↗
PubMed1999

Locations of radiation-produced DNA double strand breaks along chromosomes: a stochastic cluster process formalism.

Ionizing radiation produces DNA double strand breaks (DSBs) in chromosomes. For densely ionizing radiation, the DSBs are not spaced randomly along a chromosome: recent data for size distributions of DNA fragments indicate break clustering on kbp-Mbp scales. Different DSB clusters on a chromosome are typically made by different, statistically independent, stochastically structured radiation tracks, and the average number of tracks involved can be small. We therefore model DSB positions along a chromosome as a stationary Poisson cluster process, i.e. a stochastic process consisting of secondary point processes whose locations are determined by a primary point process that is Poisson. Each secondary process represents a break cluster, typically consisting of 1-10 DSBs in a comparatively localized stochastic pattern determined by chromatin geometry and radiation track structure. Using this Poisson cluster process model, which we call the randomly located clusters (RLC) formalism, theorems are derived for how the DNA fragment-size distribution depends on radiation dose. The RLC dose-response relations become non-linear when the dose becomes so high that DSB clusters from different tracks overlap or adjoin closely. The RLC formalism generalizes previous models, fits current data adequately and facilitates mechanistically based extrapolations from high-dose experiments to the much lower doses of interest for most applications.

Chromosome Aberrations↗
PubMed1999

Clustering of radiation-produced breaks along chromosomes: modelling the effects on chromosome aberrations.

PURPOSE: For high-LET radiations, and perhaps even for hard X-rays, DNA double-strand breaks (dsb) are clustered nonrandomly along chromosomes; disproportionately, many inter-dsb segments are less than a few Mbp (10(6) base pairs). The implications of such dsb clustering for chromosome aberrations are analysed. METHODS: Chromosome segments between different dsb within one dsb cluster are assumed too small to detect in the aberration assay. Enumeration or Monte-Carlo computer simulations are used to compute the relative frequencies of many observable aberration patterns: apparently simple or visibly complex. The theoretical predictions are compared with X-ray data for human fibroblasts, involving painted chromosomes 1, 2, 4, 5, 7 or 13. RESULTS AND CONCLUSIONS: Surprisingly, cryptic dsb multiplicity does not affect the frequency ratios predicted for aberration patterns by a random breakage-and-rejoining model. The model is generally consistent with current data on many different types of aberrations, whether or not dsb usually occur in cryptic clusters. For a Revell-type exchange model, however, the predictions do depend on clustering configurations; they gradually approach the predictions of the breakage-and-rejoining model as average cluster multiplicity increases. The model is consistent with the data, for example with the ratio of visibly complex to apparently simple aberrations, only if there is considerable dsb clustering even at low-LET, with approximately 1.5 or more reactive dsb per cluster on average.

Chromosome Aberrations↗
PubMed1998

Size distributions of misrejoining DNA fragments in irradiated cells.

When ionizing radiation strikes a cell it induces DNA double strand breaks (DSBs). Subsequently, some of the DSBs misrejoin and thus cause alterations in the size distribution of the DNA fragments. We derive a system of non-linear integro-differential equations describing the misrejoining interactions of five classes of DNA fragments, including rings and various types of linear fragments. The fragment classes are represented by density functions; the shape of a density function determines the probability that a fragment has a particular size and the amplitude (integral) equals the expected number of such fragments per cell. The equations are solved: analytically for exponentially distributed initial fragment sizes (corresponding to high doses) and numerically for arbitrary initial conditions. Computed final fragment size distributions are applied to situations representative of flow karyotypes and pulsed-field gel assays. For human flow karyotypes, the model can be used to obtain misrejoining estimates at doses too high for conventional methods of data analysis. For pulsed-field gel assays in which human chromosomes are digested with restriction endonucleases to form 'cut-somes' (restriction fragments), the model provides a means of misrejoining estimation when the cut-some sizes are non-random. The model suggests that if the cut-some size distribution for unirradiated cells is completely random, misrejoining of radiation-induced DSBs will not be detectable in the final size distribution.

Chromosome Breakage↗
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↗
PubMed1997

Intra-arm and interarm chromosome intrachanges: tools for probing the geometry and dynamics of chromatin.

Many chromosome-type, exchange-type chromosomal aberrations produced by radiation are intrachanges, i.e. involve only one chromosome. It is assumed such intrachanges are formed by illegitimate reunion of two double-strand breaks (DSBs) on the chromosome. The yield of intra-arm intrachanges (acentric rings or paracentric inversions) relative to that of interarm intrachanges (centric rings or pericentric inversions) is larger than would occur if production and illegitimate reunion of DSBs were spatially random. The excess of intra-arm intrachanges is presumably due to proximity effects for illegitimate reunions, i.e. enhancement of the intrachange probability when two DSBs are formed close to one another. Radiation track structure may also play a role. Using a polymer description for "large-scale" chromatin geometry (>2 Mb), and using two alternate (rapid or slow motion) models for the way that DSBs move after they are produced, theoretical estimates are given for size distributions of intrachanges at low or high linear energy transfer (LET). The ratio of intra-arm to interarm intrachanges is derived from the size distribution and compared with data from the literature on centric rings, inversions, interstitial deletions and excess acentric fragments. Proximity effects enhance yields of intra-arm relative to interarm intrachanges at least severalfold and perhaps as much as 10-fold compared to expectations based on spatial randomness. We argue that further measurements of intra-arm and interarm intrachanges would be informative about large-scale chromatin structure and chromosome motion. Because inversions are more frequent than estimates of randomness would indicate, and are transmissible to daughter cells, their size distribution could also help characterize past exposure to high-LET radiation.

Chromatin↗
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

Interpretation of inverse dose-rate effects for mutagenesis by sparsely ionizing radiation.

An inverse dose-rate effect has sometimes been observed for mutagenesis in cells exposed to gamma-rays. We model such data quantitatively with the key assumption that the effect is caused in cycling cells by correlated variations in sensitivity across the cell cycle, for both mutation and killing. We quantify this approach using the LQR (linear-quadratic + resensitization) formalism, which describes the response to radiation of a heterogeneous cell population. This model is applied to an exponentially growing population. We compare its predictions with dose- and dose-rate dependent mutation data and show that it can well fit the observed inverse dose-rate effect, as well as providing an explanation of why inverse dose-rate effects have been seen in some experiments, but not in others. The actual values of the model parameters emerging from the analysis are reasonable in magnitude, based on their biological interpretations. We conclude that the LQR model can quantify cell-cycle redistribution effects without overparameterization, and that the data favour a correlation explanation of inverse dose-rate effects for mutagenesis by low-LET radiation. It is less clear that this explanation is appropriate to high-LET radiation-induced oncogenic transformation, although all potential explanations of inverse dose-rate effects predict that, at appropriately low doses, no dose-rate effects of any kind are expected.

Cell Cycle↗
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↗
PubMed1995

Chromosome aberrations produced by radiation: the relationship between excess acentric fragments and dicentrics.

Most chromosome aberrations produced by ionizing radiation develop from DNA double-strand breaks (DSBs). Published data on the yield and variance of excess acentric fragments after in vitro irradiation of human lymphocytes were compared with corresponding data on dicentrics. At low LET the number of excess acentric fragments is about 60% of the number of dicentrics, independent of dose and perhaps of dose rate, suggesting that dicentrics and excess acentric fragments arise from similar kinetics rather than from fundamentally different reactions. Only a weak dependence of the ratio on LET is observed. These results are quantified using generalizations of models for pairwise DSB interactions suggested by Brewen and Brock based on data for marsupial cells. By allowing singly incomplete and some "doubly incomplete" exchanges, the models can also account for the experimental observation that the dispersion for excess acentric fragments, a measure of cell-to-cell variance, is systematically larger than the dispersion for dicentrics. Numerical estimates of an incompleteness parameter are derived.

Chromosome Aberrations↗
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↗
PubMed1994

Influence of time-dependent stochastic heterogeneity on the radiation response of a cell population.

A solid tumor is a cell population with extensive cellular heterogeneity, which severely complicates tumor treatment by therapeutic agents such as ionizing radiation. We model the response to ionizing radiation of a multicellular population whose cells have time-dependent stochastic radiosensitivity. A reaction-diffusion equation, obtained by assuming a random process with the radiation response of a cell partly determined by competition between repair and binary misrepair of DNA double-strand breaks, is used. By a suitable transformation, the equation is reduced to that of an Ornstein-Uhlenbeck process so explicit analytic solutions are available. Three consequences of the model's assumptions are that (1) response diversity within a population increases resistance to radiation, that is, the population surviving is greater than that anticipated from considering an average cell; (2) resistant cell subpopulations preferentially spared by the first part of a prolonged radiation protocol are driven biologically into more radiosensitive states as time increases, that is, resensitization occurs; (3) an inverse dose-rate effect, that is, an increase in cell killing as overall irradiation time is increased, occurs in those situations where resensitization dominates effects due to binary misrepair of repairable damage. The results are consistent with the classic results of Elkind and coworkers on extra cell killing attributed to cell-cycle redistribution and are in agreement with some recent results on in vitro and in vivo population radiosensitivity. They also generalize the therapeutic paradigm that low dose rate or fractionated radiation can help overcome hypoxic radioresistance in tumors.

Animals↗
PubMed1994

A Monte Carlo/Markov chain model for the association of data for chromosome aberrations and formation of micronuclei.

The micronucleus assay is a convenient, in situ method for observing cell damage resulting from exposure to clastogenic agents and has been widely used as a dosimeter of human exposure to radiation or chemicals. It also is a complement to the classic clonogenic cell survival assay in that it can be used to examine radiation damage vs dose as a function of cell type or radiation quality. Digitized imaging densitometry was conducted on CHO cells that have undergone one division and in which further cytokinesis was blocked to collect data on the distributions of percentage total cellular DNA per micronucleus and frequency of micronuclei per cell after gamma irradiation in G1 phase. Theoretical counterparts to both classes of distributions were generated by a Monte Carlo double-strand breakage (DSB) simulation to the CHO genome, followed by simulated repair of this initial damage using a Markov chain algorithm that assumes linear restitutions of single DSBs complete with quadratic, incomplete exchanges among pairs of DSBs. Micronuclei were presumed to consist of single acentric fragments (including fused acentric pairs). The empirical distributions, when compared to their fitted theoretical counterparts, suggest, inter alia, that: (1) a slight dependence of micronucleus size on dose exists, with a trend toward higher density in the 2-4% genome range, at the expense of the 0-2% range, with increasing dose; (2) the probability of exchange incompleteness is at least 20%; and (3) the dispersions of the micronucleus frequency distributions are progressively lower than their (essentially constant) counterparts with increasing dose. Suggested is a cooperative increase in the number of fragments per micronucleus with increasing dose. Beyond these specific results, however, it is clear that furthering the understanding of the connection between DNA aberrations and formation of micronuclei would further link these two large bodies of data.

Animals↗