Robert B. Sothern

Circadian Expression of Clock Genes in Human Oral Mucosa and Skin : Association with Specific Cell-Cycle Phases

We studied the relative RNA expression of clock genes throughout one 24-hour period in biopsies obtained from the oral mucosa and skin from eight healthy diurnally active male study participants. We found that the human clock genes hClock, hTim, hPer1, hCry1, and hBmal1 are expressed in oral mucosa and skin, with a circadian profile consistent with that found in the suprachiasmatic nuclei and the peripheral tissues of rodents. hPer1, hCry1, and hBmal1 have a rhythmic expression, peaking early in the morning, in late afternoon, and at night, respectively, whereas hClock and hTim are not rhythmic. This is the first human study to show a circadian profile of expression for all five clock genes as documented in rodents, suggesting their functional importance in man. In concurrent oral mucosa biopsies, thymidylate synthase enzyme activity, a marker for DNA synthesis, had a circadian variation with peak activity in early afternoon, coinciding with the timing of S phase in our previous study on cell-cycle timing in human oral mucosa. The major peak in hPer1 expression occurs at the same time of day as the peak in G(1) phase in oral mucosa, suggesting a possible link between the circadian clock and the mammalian cell cycle.

Circadian Variation in the Expression of Cell-Cycle Proteins in Human Oral Epithelium

At the tissue level, there is experimental and clinical data to suggest a cytokinetic coordination of the cell cycle with a greater proportion of cycling cells entering S-phase and mitosis at specific times of the day. The association of certain cell-cycle proteins with defined events in the cell cycle is well established and may be used to study the timing of cell-cycle phases over 24 hours. In this study oral mucosal biopsies were obtained from six normal human volunteers at 4-hour intervals, six times over 24 hours. Using immunohistochemistry, the number of positive cells expressing the proteins p53, cyclin-E, cyclin-A, cyclin-B1, and Ki-67 was determined for each biopsy and expressed as the number of positive cells per mm of basement membrane. We found a statistically significant circadian variation in the nuclear expression of all of these proteins with the high point of expression for p53 at 10:56 hours, cyclin-E at 14:59 hours, cyclin-A at 16:09 hours, cyclin-B1 at 21:13 hours, and Ki-67 at 02:50 hours. The circadian variation in the nuclear expression of cyclins-E (G1/S phase), -A (G2-phase), and -B1 (M-phase) with a normal physiological progression over time suggests a statistically significant circadian variation in oral epithelial cell proliferation. The finding of a circadian variation in the nuclear expression of p53 protein corresponding to late G1 is novel. This information has clinical implications regarding the timing of chemotherapy and radiotherapy.

Circadian Rhythm of Cellular Proliferation in the Human Rectal Mucosa

Circadian rhythms of DNA synthesis and cellular proliferation in the gastrointestinal mucosa have been well documented in animal models. This investigation was designed to determine whether similar rhythms could be demonstrated in the human rectal epithelium: 24 studies were performed in 16 healthy men under fasting (n = 14) and fed (n = 10) conditions. Rectal mucosal biopsy specimens were obtained through a proctoscope every 2 or 3 hours for a 24-hour span. Ex vivo measurements of tritiated thymidine incorporation into DNA were made on the mucosal samples. Feeding and time of day were each found to have an effect on the rate of thymidine incorporation into the DNA of rectal mucosal cells. Both fasted and fed subjects showed significant circadian rhythms in thymidine incorporation, which peaked at about 7 AM. Fasting lowered the overall mean thymidine uptake without altering the rhythm. Thymidine uptake generally reflects the amount of DNA synthesis in the sampled tissue. Therefore, these data may be important in the design of cancer chemotherapeutic regimens that use drugs specifically active during DNA synthesis.

Increased Tolerance of Leukemic Mice to Arabinosyl Cytosine with Schedule Adjusted to Circadian System

Mice (BDF1) inoculated with L1210 leukemia survive for a statistically significantly longer span when four courses of arabinosyl cytosine are administered at 4-day intervals-not in courses consisting of eight equal doses at 3-hour intervals, but in sinusoidally increasing and decreasing 24-hour courses, the largest amount being given at previously mapped circadian and circannual times of peak host resistance to the drug. This finding relates to the many therapeutic situations involving rhythmic, and thus predictable, cycles in the hosts tolerance of undesired effects from the agent used.

Circadian characteristics of circulating interleukin-6 in men

BACKGROUND The cytokine interleukin-6 (IL-6) is a multifunctional small-peptide molecule that is produced by various types of lymphoid and nonlymphoid cells. It plays a central role in hematopoiesis, host defense mechanisms, and acute-phase reactions, including regulation of inflammatory and immune responses. METHODS Because a circadian time structure has been shown to characterize nearly every biologic function tested in human beings, including some cytokines, we sought to investigate the 24-hour pattern of circulating IL-6 in a group of 11 clinically symptom-free men (median age, 50 years; range, 46 to 72 years). Blood samples were obtained every 3 hours for 24 hours (eight samples per subject), and serum was frozen until analysis for IL-6 with a solid-phase ELISA. RESULTS Average IL-6 values ranged from 1.66 to 5.38 pg/ml, with lowest to highest values within 24 hours ranging from 1.20 to 7.58 pg/ml (120% to 531%) between subjects. On average, values were greater than the mean throughout the night, with a peak at 01:00 hours and less than the mean throughout the day, with a nadir at 10:00 hours. A significant time effect was found by analysis of variance; and a high-amplitude circadian rhythm was described by the least-squares fit of a 24-hour cosine (p < 0.001; amplitude, 41% +/- 5%; acrophase, 02:16 +/- 00:28 hours). In addition, a positive correlation between mean IL-6 levels and age was found (r = 0.63, p = 0.037). CONCLUSIONS Because monitoring of endogenous cytokine levels is suggested for assessing immune function and pathologic condition, clinical decisions and immunotherapies may be significantly influenced by the large and predictable day-night variations in endogenous cytokine production and bioactivity.

Rhythms in human bone marrow and blood cells

In 24h studies of bone marrow (BM), circadian stage-dependent variations were demonstrated in the proliferative activity of BM cells from subsets of 35 healthy diurnally active men. On an average, the percentage of total BM cells in deoxyribonucleic acid (DNA) synthesis phase was 188% greater at midday than at midnight (circadian rhythm: p=0.018; acrophase or peak time of 13:16h). Patients with malignant disease (n=15) and a normal cortisol circadian rhythm showed higher fractions of BM cells in S-phase at midday. Colony-forming units—granulocyte/macrophage (CFU—GM), an indicator of myeloid progenitor cells, showed the same circadian variation as DNA S-phase (average range of change or ROC=136%; circadian rhythm: p<0.001; acrophase of 12:09h). Deoxyribonucleic acid S-phase and CFU—GM in BM both showed a circannual rhythm (p=0.015 and 0.008) with an identical acrophase of August 12. The daily peak in BM glutathione content, a tripeptide involved in cellular defense against cytotoxic damage, preceded BM proliferative peaks by 4–5 h (ROC=31–90%; circadian rhythm: p=0.05; acrophase of 08:30h). Myeloid (ROC=57%; circadian rhythm: p=0.056; acrophase at 08:40h) and erythroid (ROC=26%; circadian rhythm: p=0.01; acrophase of 13:01h) precursor cells were positively correlated (r=0.41; p<0.001), indicating a circadian temporal relationship and equal influence on S-phase of total BM cells. Yield of positive selected CD34+ progenitor stem cells also showed significant circadian variation (ROC=595%; circadian rhythm: p=0.02; acrophase of 12:40h). Thus, the temporal synchrony in cell cycling renders BM cells more sensitive at specific times to hematopoietic growth factors and cell cycle-specific cytotoxic drugs. Moreover, proper timing of BM harvesting may improve progenitor cell yield. When using marker rhythms in the blood to allow for individualized timing of BM procedures, the times of low values in white blood corpuscles, neutrophils, and lymphocytes and high values in cortisol were predictive of the times of highest BM erythroid, myeloid, and total S-phase numbers occurring in the following 12 h.

Transdisciplinary unifying implications of circadian findings in the 1950s

A few puzzles relating to a small fraction of my endeavors in the 1950s are summarized herein, with answers to a few questions of the Editor-in-Chief, to suggest that the rules of variability in time complement the rules of genetics as a biological variability in space. I advocate to replace truisms such as a relative constancy or homeostasis, that have served bioscience very well for very long. They were never intended, however, to lower a curtain of ignorance over everyday physiology. In raising these curtains, we unveil a range of dynamics, resolvable in the data collection and as-one-goes analysis by computers built into smaller and smaller devices, for a continued self-surveillance of the normal and for an individualized detection of the abnormal. The current medical art based on spotchecks interpreted by reference to a time-unqualified normal range can become a science of time series with tests relating to the individual in inferential statistical terms. This is already doable for the case of blood pressure, but eventually should become possible for many other variables interpreted today only based on the quicksand of clinical trials on groups. These ignore individual differences and hence the individuals needs. Chronomics (mapping time structures) with the major aim of quantifying normalcy by dynamic reference values for detecting earliest risk elevation, also yields the dividend of allowing molecular biology to focus on the normal as well as on the grossly abnormal.

Expression of clock genes Per1 and Bmal1 in total leukocytes in health and Parkinson's disease

Background:  There is a growing number of clinical studies that revealed a variety of behavioral and physiological desynchronies in patients with Parkinson’s disease (PD). However, these desynchronies have not been defined at the molecular level.

Chronobiological Analysis of Circadian Patterns in Transcription of Seven Key Clock Genes in Six Peripheral Tissues in Mice

The molecular clock machinery in mammals consists of a number of clock genes (CGs) and their resultant proteins that form interlocking transcription‐translation feedback loops. These loops generate and maintain the 24 h mRNA and protein oscillations and consequential biological and physiological rhythms. To understand whether peripheral oscillators share similarly‐timed clock machinery, the temporal expression patterns of the seven recognized key CGs (mPer1, mPer2, mCry1, mCry2, mRev‐erb alpha, mClock, and mBmal1) were examined simultaneously in six peripheral tissues in mice every 4 h for 24 h in synchronized light‐dark conditions using real time PCR assays. Time series were analyzed for time‐effect by ANOVA and for rhythm characteristics by the single cosinor fitting procedure. The expression levels of most CGs were comparable in liver, kidney, and spleen, but mBmal1 and mCry1 were more abundant in the thymus, and mPer1, mCry1, and mCry2 were more abundant in the testis. In addition, mCry2 was dramatically lower in the kidney, spleen, and thymus; mPer2 was significantly lower in the spleen, testis, and thymus; and all of the genes tested were strikingly less abundant in peripheral blood. A significant 24 h rhythmic component was found for each CG in the liver and kidney and for some CGs in other tissues. Of note, a 12 h ultradian rhythmic component was also found in mRNA expression for some CGs in several of the tissues and was the only significant oscillation observed for CGs in the testis. Ultradian oscillations were also observed for mPer1 in the testis (8 h) and thymus (12 h and 8 h) in a second study where mice were sampled every 2 h. The present results suggest that the functioning of the molecular circadian clock may be modified to some extent between peripheral tissues, as denoted by differences in amplitude and phasing, and operates differently or is less developed in tissues containing differentiating cells (i.e., testis and thymus), as denoted by the presence of ultradian patterns resulting in two or more peaks within 24 h.

Circadian Rhythmometry of Serum Interleukin-2, Interleukin-10, Tumor Necrosis Factor-α, and Granulocyte-Macrophage Colony-Stimulating Factor in Men

Serum levels of four cytokines [tumor necrosis factor-alpha (TNF-alpha), interleukin-2 (IL-2), granulocyte-macrophage colony-stimulating factor (GM-CSF), and interleukin-10 (IL-10)] were measured in nine diurnally active healthy adult male subjects at 3-h intervals during a 24-h period. Statistical evaluation by analysis of variance and/or the least- squares fit of a cosine model revealed significant 24-h rhythms for each cytokine. Although the amount of IL-2 in the serum was low, the levels fluctuated to form a single peak at approximately noon. In contrast, the other three cytokines exhibited a biphasic temporal pattern. In subjects with detectable TNF-alpha levels, the first peak occurred at 07:30 and the second at 13:30. IL-10 levels also exhibited a biphasic pattern, with one peak at 07:30 and the second 12 h later at 19:30. GM-CSF levels were last to rise, first peaking at approximately 13:30 and then again at 19:30. These results suggest temporal patterns that are unique for each cytokine, generally with daytime highs and nighttime lows.