Involvement of lipocalin 2 in leukemia and breast cancer

Human lipocalin 2 is described as the neutrophil gelatinase-associated lipocalin (NGAL). The lipocalin 2 gene encodes a small, secreted glycoprotein that possesses a variety of functions, of which the best characterized function is organic iron binding activity. Elevated NGAL expression has been observed in many human cancers including breast, colorectal, pancreatic and ovarian cancers. I focused on the characterization of NGAL function in chronic myelogenous leukemia (CML) and breast cancer. Using the leukemic xenograft mouse model, we demonstrated that over-expression of NGAL in K562 cells, a leukemic cell line, led to a higher apoptotic rate and an atrophy phenotype in the spleen of inoculated mice compared to K562 cells alone. These results indicate that NGAL plays a primary role in suppressing hematopoiesis by inducing apoptosis within normal hematopoietic cells. In the breast cancer project, we analyzed two microarray data sets of breast cancer cell lines ( n = 54) and primary breast cancer samples (n = 318), and demonstrated that high NGAL expression is significantly correlated with several tumor characteristics, including negative estrogen receptor (ER) status, positive HER2 status, high tumor grade, and lymph node metastasis. Ectopic NGAL expression in non-aggressive (ZR75.1 and MCF7) cells led to aggressive tumor phenotypes in vitro and in vivo. Conversely, knockdown of NGAL expression in various breast cancer cell lines by shRNA lentiviral infection significantly decreased migration, invasion, and metastasis activities of tumor cells both in vitro and in vivo . It has been previously reported that transgenic mice with a mutation in the region of trans-membrane domain (V664E) of HER2 develop mammary tumors that progress to lung metastasis. However, we observed that genetic deletion of the 24p3 gene, a mouse homolog of NGAL, in HER2 transgenic mice by breeding with 24p3-null mice resulted in a significant delay of mammary tumor formation and reduction of lung metastasis. Strikingly, we also found that treatment with affinity purified 24p3 antibodies in the 4T1 breast cancer mice strongly reduced lung metastasis. Our studies provide evidence that NGAL plays a critical role in breast cancer development and progression, and thus NGAL has potential as a new therapeutic target in breast cancer.^

Children with acute leukemia: A comparison of outcomes and cost-effectiveness from allogeneic blood stem cell and bone marrow transplantation

The relative merits of PBSCT versus BMT for children with standard and high risk hematologic malignancies remain unclear. In a retrospective single center study, we compared allogeneic peripheral blood stem cell transplantation (PBSCT) (n=30) with bone marrow transplantation (BMT) (n=110) in children with acute leukemia. We studied recipients of HLA matched sibling stem cells, and of stem cells from alternative donors (HLA mismatched and/or unrelated) and determined whether sourcing the stem cells from PB or marrow affected engraftment, incidence of acute and chronic GvHD, and disease-free survival at 1 year. Our results show a modest reduction in time to engraftment from PB stem cells and no greater risk of GvHD, but illustrate that the severity of the underlying disease is by far the greatest determinant of 1 year survival. Patients in the BMT group had a higher treatment success rate and lower costs than the recipients of the PBSCT within the standard but not the high risk disease group, where the treatment success rate and the cumulative costs were lower in the PBSCT group compared to the BMT group. Our current incremental cost-effectiveness ratio and analysis of uncertainty suggest that allogeneic transplantation of bone marrow grafts was a more cost-effective treatment option compared to peripheral blood stem cells in patients with standard risk childhood acute leukemia disease. For high risk disease our data are less prescriptive, since the differences were more limited and the range of costs much larger. Neither option demonstrated a clear advantage from a cost-effectiveness standpoint.^

Folate pathway polymorphisms and neuropsychological impairment after leukemia therapy

Neuropsychological impairment occurs in 20%-40% of childhood acute lymphoblastic leukemia (ALL) survivors, possibly mediated by folate depletion following methotrexate chemotherapy. We evaluated the relationship between two folate pathway polymorphisms and neuropsychological impairment after childhood ALL chemotherapy. Eighty-six childhood ALL survivors were recruited between 2004-2007 at Texas Children's Hospital after exclusion for central nervous system leukemia, cranial irradiation, and age<1 year at diagnosis. Neuropsychological evaluation at a median of 5.3 years off therapy included a parental questionnaire and the following child performance measures: Trail Making Tests A and B, Grooved Pegboard Test Dominant-Hand and Nondominant-Hand, and Digit Span subtest. We performed genotyping for polymorphisms in two folate pathway genes: reduced folate carrier (RFC1 80G>A, rs1051266) and dihydrofolate reductase (DHFR Intron-1 19bp deletion). Fisher exact test, logistic regression, Student's t-test, and ANOVA were used to compare neuropsychological test scores by genotype, using a dominant model to group genotypes. In univariate analysis, survivors with cumulative methotrexate exposure ≥9000 mg/m2 had an increased risk of attention disorder (OR=6.2, 95% CI 1.2 – 31.3), compared to survivors with methotrexate exposure <9000 mg/m2. On average, female survivors scored 8.5 points higher than males on the Digit Span subtest, a test of working memory (p=0.02). The RFC1 80G>A and DHFR Intron-1 deletion polymorphisms were not related to attention disorder or impairment on tests of attention, processing speed, fine motor speed, or memory. These data imply a strong relationship between methotrexate dose intensity and impairment in attention after childhood ALL therapy. We did not find an association between the RFC1 80G>A or DHFR Intron-1 deletion polymorphisms and long-term neuropsychological impairment in childhood ALL survivors.^

GMZ27 is a new organic arsenic derivative with anti-leukemic activity, via the induction of reactive oxygen species through NADPH oxidase and mitochondrial pathway that lead to leukemia cell apoptosis

Arsenic trioxide (ATO) is an inorganic arsenic derivative that is very effective against relapsed acute promyelocytic leukemia. It is being investigated as therapy for other cancers, but the risk/benefit ratio is questionable due to significant side effects. In contrast, organic arsenic derivatives (OAD) are known to be much less toxic than ATO. Based on high activity, we selected GMZ27 (dipropil-s-glycerol arsenic) for further study and have confirmed its potent activity against human acute leukemia cell lines. This anti-leukemic activity is significantly higher than that of ATO. Both in vivo and in vitro tests have shown that GMZ27 is significantly less toxic to normal bone marrow mononuclear cells and normal mice. Therefore, further study of the biological activity of GMZ27 was undertaken. ^ GMZ27, in contrast to ATO, can only marginally induce maturation of leukemic cells. GMZ27 has no effect on cell cycle. The anti-leukemic activity of GMZ27 against acute myeolocytic leukemia cells is not dependent upon degradation of PML-RARα fusion protein. GMZ27 causes dissipation of mitochondrial transmembrane potential, cleavage of caspase 9, caspase 3 activation. Further studies indicated that GMZ27 induces intracellular reactive oxygen species (ROS) production, and modification of intracellular ROS levels had profound effect on its potential to inhibit proliferation of leukemic cells. Therefore ROS production plays a major role in the anti-leukemic activity of GMZ27. ^ To identify how GMZ27 induces ROS, our studies focused on mitochondria and NADPH oxidase. The results indicated that the source of ROS generation induced by GMZ27 is dose dependent. At the low dose (0.3 uM) GMZ27 induces NADPH oxidase activity that leads to late ROS production, while at the high dose (2.0 uM) mitochondria function is disrupted and early ROS production is induced leading to dramatic cell apoptosis. Therefore, late, ROS production can be detected in mitochondria are depleted Rho-0 cells. Our work not only delineates a major biologic pathway for the anti-leukemic activity of GMZ27, but also discusses possible ways of enhancing the effect by the co-application of NADPH oxidase activator. Further study of this interaction may lead to achieving better therapeutic index.^

Assessment of the effect on statistical power of regression model misspecification by using techniques of mathematical statistics and simulation study

Objectives. This paper seeks to assess the effect on statistical power of regression model misspecification in a variety of situations. ^ Methods and results. The effect of misspecification in regression can be approximated by evaluating the correlation between the correct specification and the misspecification of the outcome variable (Harris 2010).In this paper, three misspecified models (linear, categorical and fractional polynomial) were considered. In the first section, the mathematical method of calculating the correlation between correct and misspecified models with simple mathematical forms was derived and demonstrated. In the second section, data from the National Health and Nutrition Examination Survey (NHANES 2007-2008) were used to examine such correlations. Our study shows that comparing to linear or categorical models, the fractional polynomial models, with the higher correlations, provided a better approximation of the true relationship, which was illustrated by LOESS regression. In the third section, we present the results of simulation studies that demonstrate overall misspecification in regression can produce marked decreases in power with small sample sizes. However, the categorical model had greatest power, ranging from 0.877 to 0.936 depending on sample size and outcome variable used. The power of fractional polynomial model was close to that of linear model, which ranged from 0.69 to 0.83, and appeared to be affected by the increased degrees of freedom of this model.^ Conclusion. Correlations between alternative model specifications can be used to provide a good approximation of the effect on statistical power of misspecification when the sample size is large. When model specifications have known simple mathematical forms, such correlations can be calculated mathematically. Actual public health data from NHANES 2007-2008 were used as examples to demonstrate the situations with unknown or complex correct model specification. Simulation of power for misspecified models confirmed the results based on correlation methods but also illustrated the effect of model degrees of freedom on power.^

BIOLOGICAL MECHANISMS AND CLINICAL IMPLICATIONS OF BCR-ABL-INDUCED MITOCHONDRIAL OXIDATIVE STRESS AND CELL SURVIVAL IN CHRONIC MYELOID LEUKEMIA

Chronic myeloid leukemia (CML), a myeloproliferative disorder, represents approximately 15-20% of all adult leukemia. The development of CML is clearly linked to the constitutively active protein-tyrosine kinase BCR-ABL, which is encoded by BCR-ABL fusion gene as the result of chromosome 9/22 translocation (Philadelphia chromosome). Previous studies have demonstrated that oxidative stress-associated genetic, metabolic and biological alterations contribute to CML cell survival and drug refractory. Mitochondria and NAD(P)H oxidase (NOX) are the major sources of BCR-ABL-induced cellular reactive oxygen species (ROS) production. However, it is still unknown how CML cells maintain the altered redox status, while escaping from the persistent oxidative stress-induced cell death. Therefore, elucidation of the mechanisms by which CML cells cope with oxidative stress will provide new insights into CML leukemogenesis. The major goal of this study is to identify the survival factors protecting CML cells against oxidative stress and develop novel therapeutic strategies to overcome drug resistance. Several experimental models were used to test CML cell redox status and cellular sensitivity to oxidative stress, including BCR-ABL inducible cell lines, BCR-ABL stably transformed cell lines and BCR-ABL-expressing CML blast crisis cells with differential BCL-XL/BCL-2 expressions. Additionally, an artificial CML cell model with heterogenic BCL-XL/BCL-2 expression was established to assess the correlation between differential survival factor expression patterns and cell sensitivity to Imatinib and oxidative stress. In this study, BCL-XL and GSH have been identified as the major survival factors responsive to BCR-ABL-promoted cellular oxidative stress and play a dominant role in regulating the threshold of oxidative stress-induced apoptosis. Cell survival factors BCL-XL and BCL-2 differentially protect mitochondria under oxidative stress. BCL-XL is an essential survival factor in preventing excessive ROS-induced cell death while BCL-2 seems to play a relatively minor role. Furthermore, the redox modulating reagent β-phenethyl isothiocyanate (PEITC) has been found to efficiently deplete GSH and induce potent cell killing effects in drug-resistant CML cells. Combination of PEITC with BCL-XL/BCL2 inhibitor ABT737 or suppression of BCL-XL by BCR-ABL inhibitor Gleevec dramatically sensitizes CML cells to apoptosis. These results have suggested that elevation of BCL-XL and cellular GSH are important for the development of CML, and that redox-directed therapy is worthy of further clinical investigations in CML.

Seasonal aspects of leukemia

An investigation of (a) month/season-of-birth as a risk factor and (b) month/season-of-treatment initation as a prognostic factor in acute lymphoblastic leukemia (ALL) in children, 0-15 years of age, was conducted. The study population used was that of the Surveillance, Epidemiology, and End Results (SEER) program of the National Cancer Institute and included children diagnosed and treated for ALL from 1973-1986. Two separate sets of analyses using different exclusion criteria led to similar results. Specifically, the inability to reject the null hypothesis of no significant difference in the variation of monthly/seasonal incidence rates among children residing within the 10 SEER sites using either cosinor analysis or one-way analysis of variance. No association was established between month/season of treatment initiation and survival in ALL among children using either Kaplan-Meier or cosinor analysis. In separate Kaplan-Meier analyses, age, gender, and treatment type were each found to be significant univariate prognostic factors for survival, however. ^

Leukemia and occupational exposure to electromagnetic fields: An incident case-control study

The existence of an association between leukemia and electromagnetic fields (EMF) is still controversial. The results of epidemiologic studies of leukemia in occupational groups with exposure to EMF are inconsistent. Weak associations have been seen in a few studies. EMF assessment is lacking in precision. Reported dose-response relationships have been based on qualitative levels of exposure to EMF without regard to duration of employment or EMF intensity on the jobs. Furthermore, potential confounding factors in the associations were not often well controlled. The current study is an analysis of the data collected from an incident case-control study. The primary objective was to test the hypothesis that occupational exposure to EMF is associated with leukemia, including total leukemia (TL), myelogenous leukemia (MYELOG) and acute non-lymphoid leukemia (ANLL). Potential confounding factors: occupational exposure to benzene, age, smoking, alcohol consumption, and previous medical radiation exposures were controlled in multivariate logistic regression models. Dose-response relationships were estimated by cumulative occupational exposure to EMF, taking into account duration of employment and EMF intensity on the jobs. In order to overcome weaknesses of most previous studies, special efforts were made to improve the precision of EMF assessment. Two definitions of EMF were used and result discrepancies using the two definitions were observed. These difference raised a question as to whether the workers at jobs with low EMF exposure should be considered as non-exposed in future studies. In addition, the current study suggested use of lifetime cumulative EMF exposure estimates to determine dose-response relationship. The analyses of the current study suggest an association between ANLL and employment at selected jobs with high EMF exposure. The existence of an association between three types of leukemia and broader categories of occupational EMF exposure, is still undetermined. If an association does exist between occupational EMF exposure and leukemia, the results of the current study suggest that EMF might only be a potential factor in the promotion of leukemia, but not its initiation. ^

Information bias and lifetime mortality risks of radiation-induced cancer

Additive and multiplicative models of relative risk were used to measure the effect of cancer misclassification and DS86 random errors on lifetime risk projections in the Life Span Study (LSS) of Hiroshima and Nagasaki atomic bomb survivors. The true number of cancer deaths in each stratum of the cancer mortality cross-classification was estimated using sufficient statistics from the EM algorithm. Average survivor doses in the strata were corrected for DS86 random error ($\sigma$ = 0.45) by use of reduction factors. Poisson regression was used to model the corrected and uncorrected mortality rates with covariates for age at-time-of-bombing, age at-time-of-death and gender. Excess risks were in good agreement with risks in RERF Report 11 (Part 2) and the BEIR-V report. Bias due to DS86 random error typically ranged from $-$15% to $-$30% for both sexes, and all sites and models. The total bias, including diagnostic misclassification, of excess risk of nonleukemia for exposure to 1 Sv from age 18 to 65 under the non-constant relative projection model was $-$37.1% for males and $-$23.3% for females. Total excess risks of leukemia under the relative projection model were biased $-$27.1% for males and $-$43.4% for females. Thus, nonleukemia risks for 1 Sv from ages 18 to 85 (DRREF = 2) increased from 1.91%/Sv to 2.68%/Sv among males and from 3.23%/Sv to 4.02%/Sv among females. Leukemia excess risks increased from 0.87%/Sv to 1.10%/Sv among males and from 0.73%/Sv to 1.04%/Sv among females. Bias was dependent on the gender, site, correction method, exposure profile and projection model considered. Future studies that use LSS data for U.S. nuclear workers may be downwardly biased if lifetime risk projections are not adjusted for random and systematic errors. (Supported by U.S. NRC Grant NRC-04-091-02.) ^

ETIOLOGIC AND CLINICAL FACTORS WHICH DIFFERENTIATE PATIENTS WITH CHROMOSOMAL ABNORMALITIES FROM DIPLOID PATIENTS IN ACUTE NONLYMPHOCYTIC LEUKEMIA

Tumor-specific chromosomal abnormalities have been demonstrated in bone marrow of approximately 50% of newly diagnosed acute nonlymphocytic (ANLL) patients. This study examined two hypotheses: (1) Aneuploid (AA) patients are diagnosed later in the course of their disease than diploid (NN) patients; and (2) AA patients are more likely to have been exposed to environmental agents. Of 324 patients eligible for study, environmental exposure data were obtained for 236 (73%) of them. No evidence was found to suggest that AA patients had more advanced disease than NN patients. Aneuploid patients were more likely than NN patients to: (a) report treatment with cytotoxic drugs for a prior medical condition (odds ratio, adjusted for age, sex and other exposures (OR) = 4.25, 95% confidence intervals, 1.38 to 13.17); (b) smoke cigarettes, OR = 1.82 (1.02, 3.26) and (c) drink alcoholic beverages, OR = 1.91 (1.05, 3.48). No statistically significant associations between aneuploidy and occupational exposures were present, OR = 3.59 (0.76, 17.13). Problems in interpreting these ORs are discussed. ^

CHARACTERIZATION OF TCL1-Tg:P53-/- MICE THAT RESEMBLE HUMAN CHRONIC LYMPHOCYTIC LEUKEMIA WITH 17P-DELETION

Chronic lymphocytic leukemia (CLL) is the most common adult leukemia in the United Statesand Europe. CLL patients with deletion of chromosome 17p, where the tumor suppressor p53 gene is located, often develop a more aggressive disease with poor clinical outcomes. However, the underlying mechanism remains unclear. In order to understand the underneath mechanism in vivo, I have recently generated mice with Eu-TCL1-Tg:p53-/- genotype and showed that these mice develop aggressive leukemia that resembles human CLL with 17p deletion. The Eu-TCL1-Tg:p53-/- mice developed CLL disease at 3-4 months, significantly earlier than the parental Eu-TCL1-Tg mice that developed CLL disease at 8-12 months. Flow cytometry analysis showed that the CD5+/ IgM+ cell population appeared in the peritoneal cavity, bone marrow, and the spleens of Eu-TCL1-Tg:p53-/- mice significantly earlier than that of the parental Eu-TCL1-Tg mice. Massive infiltration and accumulation of leukemia cells were found in the spleen and peritoneal cavity. In vitro study showed that the leukemia cells isolated from the Eu-TCL1-Tg:p53-/- mice were more resistant to fludarabine treatment than the leukemia cells isolated from spleens of Eu-TCL1-Tg mice. Interestingly, TUNEL assay revealed that there was higher apoptotic cell death found in the Eu-TCL1-Tg spleen tissue compared to the spleens of the Eu-TCL1-Tg:p53-/- mice, suggesting that the loss of p53 compromises the apoptotic process in vivo, and this might in part explain the drug resistant phenotype of CLL cells with 17p-deletion. In the present study, we further demonstrated that the p53 deficiency in the TCL1 transgenic mice resulted in significant down-regulation of microRNAs miR-15a and miR16-1, associated with a substantial up-regulation of Mcl-1, suggesting that the p53-miR15a/16-Mcl-1 axis may play an important role in CLL pathogenesis. Interestingly, we also found that loss of p53 resulted in a significant decrease in expression of the miR-30 family especially miR-30d in leukemia lymphocytes from the Eu-TCL1-Tg:p53-/- mice. Such down-regulation of those microRNAs and up-regulation of Mcl-1 were also found in primary leukemia cells from CLL patients with 17p deletion. To further exam the biological significance of decrease in the miR-30 family in CLL, we investigated the potential involvement of EZH2 (enhancer of zeste homolog 2), a component of the Polycomb repressive complex known to be a downstream target of miR-30d and plays a role in disease progression in several solid cancers. RT-PCR and western blot analyses showed that both EZH2 mRNA transcript and protein levels were significantly increased in the lymphocytes of Eu-TCL1-Tg:p53-/- mice relative to Eu-TCL1-Tg mice. Exposure of leukemia cells isolated from Eu-TCL1-Tg:p53-/- mice to the EZH2 inhibitor 3-deazaneplanocin (DZNep) led to induction of apoptosis, suggesting EZH2 may play a role in promoting CLL cell survival and this may contribute to the aggressive phenotype of CLL with loss of p53. Our study has created a novel CLL mouse model, and suggests that the p53/miR15a/16-Mcl-1 axis & p53/miR30d-EZH2 may contribute to the aggressive phenotype and drug resistance in CLL cells with loss of p53.

Molecular analysis of a neurovirulent murine leukemia virus, {\it ts\/}1

ts1 is a neurovirulent spontaneous temperature-sensitive mutant of Moloney murine leukemia virus TB (MoMuLV-TB). MoMuLV-TB causes T-cell lymphoma or lymphoid leukemia in mice after a long latency period whereas ts1 causes a progressive hindlimb paralytic disease after a much shorter latency period. In previous studies, it had been shown that the temperature-sensitive defect resided in the $env$ gene. At the restrictive temperature, the envelope precursor polyprotein, gPr80$\sp{env}$, is inefficiently processed intracellularly into a heterodimer consisting of two cleavage products, gp70 and Prp15E. This inefficient processing is correlated with neurovirulence. In this study, the nucleotide sequences of the env genes for both ts1 and MoMuLV-TB were determined, and the encoded amino acid sequences were deduced from the DNA sequences. There were four unique amino acid substitutions in the gPr80$\sp{env}$ of ts1. In order to determine which unique amino acid was responsible for the phenotypic characteristics of ts1, a set of hybrid genomes was constructed by exchanging restriction fragments between ts1 and MoMuLV-TB. NIH 3T3 cells were transfected with the hybrid genomes to obtain infectious hybrid viruses. Assays of the hybrid viruses showed that a Val-25$\to$Ile substitution in gPr80$\sp{env}$ was responsible for the temperature sensitivity, inefficient processing, and neurovirulence of ts1. In further studies, the Ile-25 in gPr80$\sp{env}$ was substituted with Thr, Ala, Leu, Gly, and Glu by site-directed mutagenesis to generate a new set of mutant viruses, i.e., ts1-T, -A, -L, -G, and -E, respectively. The rank order of the mutants for temperature sensitivity was: ts1-E $>$ ts1-G $>$ ts1-L $>$ ts1-A $>$ ts1 $>$ ts1-T. The degree of temperature sensitivity of each of the mutants also correlated with the degree of inefficient processing of gPr80$\sp{env}$. The mutant viruses were assayed for neurovirulence. ts1-T caused whole body tremor, ts1-A caused hindlimb paralysis, ts1-L caused paraparesis, but ts1-G and -E were not neurovirulent. These results show that inefficient processing of gPr80$\sp{env}$ is correlated with neurovirulence, but if processing of gPr80$\sp{env}$ is too inefficient there is no neurovirulence. Furthermore, the disease profile of each of the neurovirulent viruses depends on the degree of inefficient processing of gPr80$\sp{env}$. ^

Statistics of the National soil test database of France

In France, farmers commission about 250,000 soil-testing analyses per year to assist them managing soil fertility. The number and diversity of origin of the samples make these analyses an interesting and original information source regarding cultivated topsoil variability. Moreover, these analyses relate to several parameters strongly influenced by human activity (macronutrient contents, pH...), for which existing cartographic information is not very relevant. Compiling the results of these analyses into a database makes it possible to re-use these data within both a national and temporal framework. A database compilation relating to data collected over the period 1990-2009 has been recently achieved. So far, commercial soil-testing laboratories approved by the Ministry of Agriculture have provided analytical results from more than 2,000,000 samples. After the initial quality control stage, analytical results from more than 1,900,000 samples were available in the database. The anonymity of the landholders seeking soil analyses is perfectly preserved, as the only identifying information stored is the location of the nearest administrative city to the sample site. We present in this dataset a set of statistical parameters of the spatial distributions for several agronomic soil properties. These statistical parameters are calculated for 4 different nested spatial entities (administrative areas: e.g. regions, departments, counties and agricultural areas) and for 4 time periods (1990-1994, 1995-1999, 2000-2004, 2005-2009). Two kinds of agronomic soil properties are available: the firs one correspond to the quantitative variables like the organic carbon content and the second one corresponds to the qualitative variables like the texture class. For each spatial unit and temporal period, we calculated the following statistics stets: the first set is calculated for the quantitative variables and corresponds to the number of samples, the mean, the standard deviation and, the 2-,4-,10-quantiles; the second set is calculated for the qualitative variables and corresponds to the number of samples, the value of the dominant class, the number of samples of the dominant class, the second dominant class, the number of samples of the second dominant class.

Path characteristics and colony arrival-departure statistics of king penguins (Aptenodytes patagonicus) on Possession and Kerguelen Island

Orientation based on visual cues can be extremely difficult in crowded bird colonies due to the presence of many individuals. We studied king penguins (Aptenodytes patagonicus) that live in dense colonies and are constantly faced with such problems. Our aims were to describe adult penguin homing paths on land and to test whether visual cues are important for their orientation in the colony. We also tested the hypothesis that older penguins should be better able to cope with limited visual cues due to their greater experience. We collected and examined GPS paths of homing penguins. In addition, we analyzed 8 months of penguin arrivals to and departures from the colony using data from an automatic identification system. We found that birds rearing chicks did not minimize their traveling time on land and did not proceed to their young (located in creches) along straight paths. Moreover, breeding birds' arrivals and departures were affected by the time of day and luminosity levels. Our data suggest that king penguins prefer to move in and out of the colony when visual cues are available. Still, they are capable of navigating even in complete darkness, and this ability seems to develop over the years, with older breeding birds more likely to move through the colony at nighttime luminosity levels. This study is the first step in unveiling the mysteries of king penguin orientation on land.