Evolution of resistance during clonal expansion

Acquired drug resistance is a major limitation for cancer therapy. Often, one genetic alteration suffices to confer resistance to an otherwise successful therapy. However, little is known about the dynamics of the emergence of resistant tumor cells. In this article, we consider an exponentially growing population starting from one cancer cell that is sensitive to therapy. Sensitive cancer cells can mutate into resistant ones, which have relative fitness alpha prior to therapy. In the special case of no cell death, our model converges to the one investigated by Luria and Delbrück. We calculate the probability of resistance and the mean number of resistant cells once the cancer has reached detection size M. The probability of resistance is an increasing function of the detection size M times the mutation rate u. If Mu < 1, then the expected number of resistant cells in cancers with resistance is independent of the mutation rate u and increases with M in proportion to M(1-1/alpha) for advantageous mutants with relative fitness alpha>1, to l nM for neutral mutants (alpha = 1), but converges to an upper limit for deleterious mutants (alpha<1). Further, the probability of resistance and the average number of resistant cells increase with the number of cell divisions in the history of the tumor. Hence a tumor subject to high rates of apoptosis will show a higher incidence of resistance than expected on its detection size only.     

Linoleic acid-grafted chitosan oligosaccharide micelles for intracellular drug delivery and reverse drug resistance of tumor cells

Intracellular drug delivery is an important rout to reverse drug resistance of tumor cells. In this study, the linoleic acid (LA)-grafted chitosan oligosaccharide (CSO) was synthesized to construct a micellar delivery system for intracellular delivery. The synthesized linoleic acid-grafted chitosan oligosaccharide (CSO-LA) with 10.3% graft ratio of LA could form micelles in aqueous with 86.69 μg/ml critical micellar concentration (CMC). The CSO-LA micelle had 46.2±3.6 nm number average diameter and 36.0±3.3 mV zeta potential. Taking doxorubicin base (DOX) as a model drug, the drug-loaded CSO-LA micelles (CSO-LA/DOX) was then prepared. The drug encapsulation efficiencies of CSO-LA/DOX were as high as 80%, and the drug loading capacity could be improved by increasing the charged DOX. Using MCF-7, Doxorubicin·HCl resistant MCF-7 (MCF-7/ADR), K562 and Doxorubicin·HCl resistant K562 (K562/ADR) cells as model drug sensitive and drug resistant tumor cells, the experiments demonstrated the CSO-LA had excellent cellular uptake ability by either drug sensitive tumor cells or drug resistance tumor cells. The CSO-LA micelles could deliver DOX into tumor cells, and the DOX in cells was increased with incubation time. As a result, the cytotoxicities of DOX encapsulated in CSO-LA micelles against drug resistance tumor cells were improved significantly, comparing to that of Doxorubicin·HCl solution.     

Stochastic modeling of drug resistance in cancer

One of the main causes of failure in the treatment of cancer is the development of drug resistance by the cancer cells. Employing multi-drug therapeutic strategies is a promising way to prevent resistance and improve the chances of treatment success. We formulate and analyse a stochastic model for multi-drug resistance and investigate the dependence of treatment outcomes on the initial tumor load, mutation rates and the turnover rate of cancerous cells. We elucidate the general principles of the emergence and evolution of resistant cells inside the tumor, before and after the start of treatment. We discover that for non-mutagenic drugs, pre-existence contributes more to resistance generation than the treatment phase; this result holds for the case where all drugs are applied simultaneously, and is not applicable for sequential therapy models. The application of mathematical modelling to aspects of adjuvant chemotherapy scheduling. J. Math. Biol. 48(4), 375-422]. Also, we find that treatment success is independent on the turnover rate for one drug, and it depends strongly on it for multi-drug therapies. For low-turnover rates, increasing the number of drugs will increase the probability of successful therapy. For very high-turnover rates, increasing the number of drugs used does not significantly increase the chances of treatment success.     

Priming cancer cells for drug resistance: role of the fibroblast niche

Conventional and targeted chemotherapies remain integral strategies to treat solid tumors. Despite the large number of anti-cancer drugs available, chemotherapy does not completely eradicate disease. Disease recurrence and the growth of drug resistant tumors remain significant problems in anti-cancer treatment. To develop more effective treatment strategies, it is important to understand the underlying cellular and molecular mechanisms of drug resistance. It is generally accepted that cancer cells do not function alone, but evolve through interactions with the surrounding tumor microenvironment. As key cellular components of the tumor microenvironment, fibroblasts regulate the growth and progression of many solid tumors. Emerging studies demonstrate that fibroblasts secrete a multitude of factors that enable cancer cells to become drug resistant. This review will explore how fibroblast secretion of soluble factors act on cancer cells to enhance cancer cell survival and cancer stem cell renewal, contributing to the development of drug resistant cancer.     

Intracellular distribution of anthracyclines in drug resistant cells

The unresponsiveness of multidrug resistant tumor cells to antineoplastic chemotherapy is often associated with reduced cellular drug accumulation accomplished by overexpressed transport molecules. Moreover, intracellular drug distribution in resistant cells appears to be remarkably different when compared to their wild type counterparts. In the present paper, we report observations on the intracellular accumulation and distribution of doxorubicin, an antitumoral agent widely employed in chemotherapy, in sensitive and resistant cultured tumor cells. The inherent fluorescence of doxorubicin allowed us to follow its fate in living cells by laser scanning confocal microscopy. This study included flow cytometric analysis of drug uptake and efflux and analysis of the presence of the well known drug transporter P-glycoprotein. Morphological, immunocytochemical and functional data evidentiated the Golgi apparatus as the preferential intracytoplasmic site of drug accumulation in resistant cells, capable of sequestering doxorubicin away from the nuclear target. Moreover, P-glycoprotein has been found located in the Golgi apparatus in drug induced resistant cells and in intrinsic resistant cells, such as melanoma cells. Thus, this organelle seems to play a pivotal role in the intracellular distribution of doxorubicin. This revised version was published online in August 2006 with corrections to the Cover Date.     

Stochastic modeling of cellular colonies with quiescence: an application to drug resistance in cancer

Several cancers are thought to be driven by cells with stem cell like properties. An important characteristic of stem cells, which also applies to primitive tumor cells, is the ability to undergo quiescence, where cells can temporarily stop the cell cycle. Cellular quiescence can affect the kinetics of tumor growth, and the susceptibility of the cells to therapy. To study how quiescence affects treatment, we formulate a stochastic birth-death process with quiescence, on a combinatorial cellular mutation network, and consider the pre-treatment (growth) and treatment (decay) regimes. We find that, in the absence of mutations, treatment (if sufficiently strong) will proceed as a biphasic decline with the first (faster) phase driven by the elimination of the cycling cells and the second (slower) phase limited by the process of cell awakening. Other regimes are possible for weaker treatments. We also describe how the process of mutant generation is influenced by quiescence. Interestingly, for single-drug treatments, the probability to have resistance at start of treatment is independent of quiescence. For two or more drugs, the probability to have generated resistant mutants before treatment grows with quiescence. Finally, we study the influence of quiescence on the treatment phase. Starting from a given composition of mutants, the chances of treatment success are not influenced by the presence of quiescence.     

Stochastic modeling of cellular colonies with quiescence: An application to drug resistance in cancer

Several cancers are thought to be driven by cells with stem cell like properties. An important characteristic of stem cells, which also applies to primitive tumor cells, is the ability to undergo quiescence, where cells can temporarily stop the cell cycle. Cellular quiescence can affect the kinetics of tumor growth, and the susceptibility of the cells to therapy. To study how quiescence affects treatment, we formulate a stochastic birth–death process with quiescence, on a combinatorial cellular mutation network, and consider the pre-treatment (growth) and treatment (decay) regimes. We find that, in the absence of mutations, treatment (if sufficiently strong) will proceed as a biphasic decline with the first (faster) phase driven by the elimination of the cycling cells and the second (slower) phase limited by the process of cell awakening. Other regimes are possible for weaker treatments. We also describe how the process of mutant generation is influenced by quiescence. Interestingly, for single-drug treatments, the probability to have resistance at start of treatment is independent of quiescence. For two or more drugs, the probability to have generated resistant mutants before treatment grows with quiescence. Finally, we study the influence of quiescence on the treatment phase. Starting from a given composition of mutants, the chances of treatment success are not influenced by the presence of quiescence.     

The effect of cellular differentiation on the development of permanent drug resistance

A stem cell compartment model is utilized to simulate the growth of human tumors. This model is used to explore the effect of cell differentiation and loss on the development of spontaneous drug resistance. Cellular differentiation is found to increase the rate of development of single drug resistance, although this is balanced by the likehood that such resistant cells will subsequently become extinct. Overall the probability that singly resistant cells will develop and persist is found to be independent of the rate of cellular differentiation. Conversely, when two drugs are available, the probability that cells resistant to both drugs will persist is proportional to the rate of cellular differentiation. Approximate formulae relating the net overall mutation rate to the intrinsic mutation rates and net growth rates of the stem cell compartment are developed.     

Multiple drug resistance of tumor cells: manifestations, genetic basis, clinical aspects

Data are reviewed concerning the results of study of multidrug-resistant (MDR) tumor cells. MDR often develops in the course of chemotherapy or in vitro selection of tumor cells by vincristine, adriamycin, actinomycin D, colchicine, etc. MDR cells are resistant to all these drugs though their targets and mechanisms of toxic action are quite different. Resistance is due to the decreased accumulation by MDR cells of these compounds. The genetic basis for MDR is amplification of a large genomic region that contains a number of genes coding for products and functions that are under extensive study. Specific karyotype and amplified DNA alterations occur during the development of MDR imitating the processes of appearance and variability of multigene families. The obtained data demonstrate the ways of overcoming of tumor multidrug resistance in clinic.     

Probability of resistance evolution for exponentially growing virus in the host

Chemotherapy for tumor and pathogenic virus often faces an emergence of resistant mutants, which may lead to medication failure. Here we study the risk of resistance to evolve in a virus population which grows exponentially. We assume that infected cells experience a "proliferation event" of virus at a random time and that the number of newly infected cells from an infected cell follows a Poisson distribution. Virus starts from a single infected cell and the virus infection is detected when the number of infected cells reaches a detection size. Initially virus is sensitive to a drug but later acquires resistance by mutations. We ask the probability that one or more cells infected with drug-resistant virus exist at the time of detection. We derive a formula for the probability of resistance and confirm its accuracy by direct computer simulations. The probability of resistance increases with detection size and mutation rate but decreases with the population growth rate of sensitive virus. The risk of resistance is smaller when more cells are newly infected by viral particles from a single infected cell if the viral growth rate is the same.     

Pre-existence and emergence of drug resistance in HIV-1 infection.

Antiviral treatment of HIV-1 infection often fails because of the rapid emergence of resistant virus within weeks of the start of therapy. This raises the question of whether resistant viruses pre-exist in drug-naive patients or whether it is produced after the start of therapy. Here we compare the likelihood of pre-existence with the likelihood of production of resistant virus during therapy. We show that provided resistant virus pre-exists, then a stronger therapy may lead to a greater initial reduction of virus load, but will also cause a faster rise of resistant virus. In this case the total benefit of treatment is independent of the degree of inhibition of sensitive virus. If, on the other hand, resistant mutants do not pre-exist, then the emergence of resistance during treatment depends on the efficacy of the drug. If the drug is sufficiently potent to eradicate sensitive virus, then the probability that resistant mutants first appear during therapy is smaller than the probability that they existed before therapy. If the drug cannot eradicate the sensitive virus, then after sufficiently long time resistant mutants will appear. However, mutants that are unlikely to pre-exist may taken long time to appear.     

Evaluation of tumor heterogeneity of prostate carcinoma by flow- and image DNA cytometry and histopathological grading.

BACKGROUND: Heterogeneity of prostate carcinoma is one of the reasons for pretreatment underestimation of tumor aggressiveness. We studied tumor heterogeneity and the probability of finding the highest tumor grade and DNA aneuploidy with relation to the number of biopsies. MATERIAL AND METHODS: Specimens simulating core biopsies from five randomly selected tumor areas from each of 16 B?cking's grade II and 23 grade III prostate carcinomas were analyzed for tumor grade and DNA ploidy by flow- and fluorescence image cytometry (FCM, FICM). Cell cycle composition was measured by FCM. RESULTS: By determination of ploidy and cell cycle composition, morphologically defined tumors can further be subdivided. Heterogeneity of tumor grade and DNA ploidy (FCM) was 54% and 50%. Coexistence of diploid tumor cells in aneuploid specimens represents another form of tumor heterogeneity. The proportion of diploid tumor cells decreased significantly with tumor grade and with increase in the fraction of proliferating cell of the aneuploid tumor part. The probability of estimating the highest tumor grade or aneuploidy increased from 40% for one biopsy to 95% for 5 biopsies studied. By combining the tumor grade with DNA ploidy, the probability of detecting a highly aggressive tumor increased from 40% to 70% and 90% for one and two biopsies, respectively. CONCLUSION: Specimens of the size of core biopsies can be used for evaluation of DNA ploidy and cell cycle composition. Underestimation of aggressiveness of prostate carcinoma due to tumor heterogeneity is minimized by simultaneous study of the tumor grade and DNA ploidy more than by increasing the number of biopsies. The biological significance of coexistent diploid tumor cell in aneuploid lesions remains to be evaluated.     

Modulation of drug resistance in homoharringtonine-resistant C-1300 neuroblastoma cells with cyclosporine A and dipyridamole.

The development of resistance accounts for therapy failure in the majority of advanced cases of neuroblastoma in children. A new transplantable murine C-1300 neuroblastoma cell line was developed in vitro, by repeated exposure of a sensitive cell line to increasing, but sublethal, doses of Homoharringtonine (HHT). The ED50 of the highly resistant cells for HHT, using a standard agar colony assay, is 480 ng/ml, compared with 13 ng/ml for the sensitive parental line. The resistant cells have cross-resistance to a number of other agents, including adriamycin, vinca alkaloids, melphalan, and CCNU. Western blot analysis revealed progressive increases in P-glycoprotein, parallel to the graded development of resistance with a 29-fold elevation in the highest resistant cells. High-performance liquid chromatography (HPLC) indicated that resistant cells have a significantly lower uptake of HHT than parental sensitive cells. cyclosporine A (CsA) and dipyridamole (DPM) could modulate the acquired resistance and completely restore the cytotoxic effects of HHT and adriamycin as determined by the clonogenic assay. The reversal of resistance by CsA and DPM was dose dependent. With the relative low toxicity of dipyridamole and CsA in doses required for modulation of resistance, these agents may be candidates for clinical utilization in chemotherapy of resistant neuroblastoma.     

Interaction between daunorubicin and chromatin from Ehrlich ascites tumor cells.

Interaction of daunorubicin with chromatin from Ehrlich ascites tumor cells has been studied by spectrofluorimetry. Daunorubicin interacts with chromatin and displays at least two types of binding. The number of binding sites is reduced when compared to deoxyribonucleic acid. There is no difference in the overall structure of chromatins extracted from cells sensitive or resistant to daunorubicin.     

Suppression of growth of guinea pig line-10 hepatocarcinoma. I. Effect of simultaneous administration of tumor cells and antibodies from normal rabbits.

Suppression of growth of the line-10 hepatocarcinoma in strain-2 guinea pigs occurred when line-10 cells were injected intradermally together with sera or immunoglobulins derived from normal rabbits. A significant number of animals were resistant to subsequent rechallenge with tumor cells. This immunity was specific, depended on contact of immunoglobulins with tumor cells and on the concentration of immunoglobulins. Repeated injections acted as potent vaccines and resulted in the development of immunity in 84.6% of recipients. Fc receptors were not detected on line-10 cells. Antibodies reacting with line-10 cell unique antigens as well as with antigens common to line-10, line-1 and normal guinea pig spleen cells were found in NRS. Injection of line-10 cells together with rabbit immunoglobulins from which antibodies reacting with antigens derived from line-10 cells had been removed did not result in tumor suppression. The specific antigen(s) recognized by antibodies that suppressed growth of the line-10 tumor in vivo was not determined.     

Mathematical Models of Gene Amplification with Applications to Cellular Drug Resistance and Tumorigenicity

An increased number of copies of specific genes may offer an advantage to cells when they grow in restrictive conditions such as in the presence of toxic drugs, or in a tumor. Three mathematical models of gene amplification and deamplification are proposed to describe the kinetics of unstable phenotypes of cells with amplified genes. The models differ in details but all assume probabilistic mechanisms of increase and decrease in gene copy number per cell (gene amplification/deamplification). Analysis of the models indicates that a stable distribution of numbers of copies of genes per cell, observed experimentally, exists only if the probability of deamplification exceeds the probability of amplification. The models are fitted to published data on the loss of methotrexate resistance in cultured cell lines, due to the loss of amplified dihydrofolate reductase gene. For two mouse cell lines unstably resistant to methotrexate the probabilities of amplification and deamplification of the dihydrofolate reductase gene on double minute chromosomes are estimated to be approximately 2% and 10%, respectively. These probabilities are much higher than widely presumed. The models explain the gradual disappearance of the resistant phenotype when selective pressure is withdrawn, by postulating that the rate of deamplification exceeds the rate of amplification. Thus it is not necessary to invoke a growth advantage of nonresistant cells which has been the standard explanation. For another analogous process, the loss of double minute chromosomes containing the myc oncogene from SEWA tumor cells, the growth advantage model does seem to be superior to the amplification and deamplification model. In a more theoretical section of the paper, it is demonstrated that gene amplification/deamplification can result in reduction to homozygosity, such as is observed in some tumors. Other applications are discussed.     

Interaction between radiation and drug damage in mammalian cells. IV. Radiation response of adriamycin-resistant V79 cells

Adriamycin-resistant variants derived from V79 Chinese hamster cells were examined for their radiation response properties. A stable resistant cell line (77A) demonstrated a significant reduction in the extrapolation number of the single-dose radiation survival curve. Second-step mutants from 77A cells exhibited a spectrum of radiation response states including decreased D0 values and large extrapolation numbers. A highly Adriamycin-resistant line (LZ) was found to be radiation sensitive with increased capacity for the accumulation of sublethal radiation injury. LZ cells are known to contain double-minute chromosomes and an amplified gene for the multidrug phenotype and to exhibit multidrug resistant properties. These cells require the presence of Adriamycin in their growth medium to maintain their pleiotropic characteristics. LZ cells became more resistant to radiation following reversion to an intermediate Adriamycin response as the consequence of growth in Adriamycin-free medium. Reverted cells also lost their large capacity for sublethal damage. It is suggested that detailed study of these mutants may provide insight into the identification of radiation-sensitive sites and their relationship to the genetic changes characterizing Adriamycin-resistant cell lines.     

The probability of treatment induced drug resistance

We propose a discrete time branching process to model the appearance of drug resistance under treatment. Under our assumptions at every discrete time a pathogen may die with probability 1−p or divide in two with probability p. Each newborn pathogen is drug resistant with probability μ. We start with N drug sensitive pathogens and with no drug resistant pathogens. We declare the treatment successful if all pathogens are eradicated before drug resistance appears. The model predicts that success is possible only if p<1/2. Even in this case the probability of success decreases exponentially with the parameter m=μN. In particular, even with a very potent drug (i.e. p very small) drug resistance is likely if m is large.     

The anticancer drug,cisplatin, increases the naturally occurring cell-mediated lysis of tumor cells

Summary It has been proposed that a component of the antitumor potential of the chemotherapeutic agent, cisplatin, resides in the host's ability to respond to cisplatintreated tumor cells. Here we report that tumor cells that are normally resistant to lysis mediated by naturally occurring cytotoxic cells showed an increased sensitivity to lysis mediated by murine spleen cells or human peripheral blood monocytes and lymphocytes when cisplatin was added at the beginning of the lytic assay. This was shown for the lysis of both murine and human tumor cells. The pretreatment of tumor cells, but not effector cells with cisplatin caused an increase in lysis in the presence of murine spleen cells or human peripheral blood leukocytes, indicating that the effect of cisplatin is to reduce resistance to lysis by these effector cells. The lysis of tumor cells by naturally occurring cytotoxic cells was blocked by antibodies specific for tumor necrosis factor. In addition, the ability of cisplatin to increase lysis was seen with cells that are sensitive to natural cytotoxic cells, but not with cells that are sensitive to natural killer cells. These results suggest that the effector cells that mediate the lysis of these tumor cells in the presence of cisplatin are likely to be natural cytotoxic cells. The ability of cisplatin to increase the lysis of tumor cells by naturally occurring cytotoxic cells indicates that these cells may be a host defense mechanism that contributes to the anticancer potential of cisplatin.     

Imatinib interferes with survival of multi drug resistant Kaposi's sarcoma cells

Multi drug resistance (MDR) is defined as the ability of tumor cells to become resistant to unrelated drugs. Tyrosine kinase inhibitor imatinib has been demonstrated to be effective in the treatment of certain tumors. In particular, imatinib inhibits Bcr-Abl kinase activity, c-kit and the phosphorylation of platelet-derived growth factor (PDGF) receptors. In this work, we show that imatinib inhibits PDGF phosphorylation not only in wt Kaposi sarcoma (KS) but also in multi drug resistant KS cells. This was associated with an increased apoptosis in wt cells and an increased autophagy in MDR-KS cells. These data add new insights to the possible use of imatinib in the overcoming of MDR in KS cells.