William J. M. Hrushesky

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.

The effects of surgery on tumor growth: a century of investigations

A few clinical investigations suggest that while primary breast cancer surgical removal favorably modifies the natural history for some patients, it may also hasten the metastatic development for others. The concepts underlying this disease paradigm, i.e. tumor homeostasis, tumor dormancy and surgery-driven enhancement of metastasis development, have a long history that is reviewed. The review reveals the context in which these concepts were conceived and structured to explain experimental data and shows that they are not so new and far fetched. The idea that surgical cancer resection has both beneficial and adverse effects upon cancer spread and growth that result from the modulation of tumor dormancy by the resection should be considered a potentially fruitful working hypothesis.

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.

Tumor dormancy and surgery-driven interruption of dormancy in breast cancer: learning from failures

Primary tumor removal, usually considered intrinsically beneficial, can perturb metastatic homeostasis, and for some patients results in the acceleration of metastatic cancer. The continuous-growth model is required to yield to an interrupted-growth model, the implications of which are episodes of tumor dormancy. This Review analyzes the recent evolution of two paradigms related to the development of breast cancer metastases. The evolution of the paradigms described herein is supported by a growing body of findings from experimental models, and is required to explain breast cancer recurrence dynamics for patients undergoing surgery with or without adjuvant chemotherapy.

Circadian-shaped infusions of floxuridine for progressive metastatic renal cell carcinoma.

Sixty-eight unselected patients with progressive metastatic renal cell carcinoma (RCC) were treated between March 1985 and November 1988 with continuous infusion floxuridine (FUDR). Thirty-seven percent of these patients had previously received and failed systemic treatment. Using implantable pumps for automatic drug delivery, FUDR was continuously infused for 14 days at monthly intervals. The starting dose was 0.15 mg/kg/d (intravenous [IV]; n = 61) or 0.25 mg/kg/d (intraarterial [IA]; n = 7); IV doses were increased or decreased in increments of 0.025 mg/kg/d as permitted by toxicity. Diarrhea (with or without mild abdominal cramping) and nausea/vomiting limited the FUDR IV infusion, and hepatic function abnormalities limited FUDR IA infusion. The use of a circadian-modified infusion schedule permitted high FUDR doses to be safely given as compared with a constant rate infusion schedule. Of 63 patients assessable for response, 56 received systemic FUDR infusion. Four complete responses (CRs; 7.1%); and seven partial responses (PRs; 12.5%) were observed (objective response rate, CR plus PR, 19.6 +/- 5.1% [95% confidence limits] ). The median objective response duration was 10.8 months (range, 1 to 18 months; mean, 9.4 +/- 1.6). Four additional patients had minor tumor responses (MRs; 7.1%). In a subgroup of seven assessable patients receiving hepatic arterial FUDR, we observed one CR and three PRs (57.2 +/- 42.8%). Overall, objective response (CR plus PR) was seen in a quarter of assessable patients treated, 15 of 63, while only 15 of the 63 assessable patients (25.4%) have had objective tumor progression. The median follow-up time for all 68 patients was 28 months (range, 1 to 42), and their median survival duration is 15 months (range, 3 to 37 months). Continuous infusion FUDR is an effective outpatient treatment for progressive metastatic RCC, producing durable tumor response and causing little toxicity.

Period 2 mutation accelerates ApcMin/+ tumorigenesis.

Colorectal cancer risk is increased in shift workers with presumed circadian disruption. Intestinal epithelial cell proliferation is gated throughout each day by the circadian clock. Period 2 (Per2) is a key circadian clock gene. Per2 mutant (Per2m/m) mice show an increase in lymphomas and deregulated expression of cyclin D and c-Myc genes that are key to proliferation control. We asked whether Per2 clock gene inactivation would accelerate intestinal and colonic tumorigenesis. The effects of PER2 on cell proliferation and β-catenin were studied in colon cancer cell lines by its down-regulation following RNA interference. The effects of Per2 inactivation in vivo on β-catenin and on intestinal and colonic polyp formation were studied in mice with Per2 mutation alone and in combination with an Apc mutation using polyp-prone ApcMin/+ mice. Down-regulation of PER2 in colon cell lines (HCT116 and SW480) increases β-catenin, cyclin D, and cell proliferation. Down-regulation of β-catenin along with Per2 blocks the increase in cyclin D and cell proliferation. Per2m/m mice develop colonic polyps and show an increase in small intestinal mucosa β-catenin and cyclin D protein levels compared with wild-type mice. ApcMin/+Per2m/m mice develop twice the number of small intestinal and colonic polyps, with more severe anemia and splenomegaly, compared with ApcMin/+ mice. These data suggest that Per2 gene product suppresses tumorigenesis in the small intestine and colon by down-regulation of β-catenin and β-catenin target genes, and this circadian core clock gene may represent a novel target for colorectal cancer prevention and control. (Mol Cancer Res 2008;6(11):1786–93)

Dormancy and surgery-driven escape from dormancy help explain some clinical features of breast cancer

To explain bimodal relapse patterns observed in breast cancer data, we have proposed that metastatic breast cancer growth commonly includes periods of temporary dormancy at both the single cell phase and the avascular micrometastasis phase. The half‐lives of these states are 1 and 2 years respectively. We also suggested that surgery to remove the primary tumor often terminates dormancy resulting in accelerated relapses. These iatrogenic events are very common in that over half of all metastatic relapses progress in that manner. Assuming this is true, there should be ample and clear evidence in clinical data. We review here the breast cancer paradigm from early detection, through treatment and follow‐up, and consider how dormancy and surgery‐driven escape from dormancy would be observed. We examine mammography data, effectiveness of adjuvant chemotherapy, heterogeneity and aggressiveness, timing of surgery within the menstrual cycle and racial differences in outcome. Dormancy can be identified in these diverse data but most conspicuous is the sudden escape from dormancy following primary surgery. These quantitative findings provide linkage between experimental studies of tumor dormancy and clinical efforts to improve patient outcome.

Circadian clock coordinates cancer cell cycle progression, thymidylate synthase, and 5-fluorouracil therapeutic index

Dysregulated cellular proliferation is a characteristic property of cancer. We show that, despite this fact, cancers maintain high amplitude, circadian rhythms in their growth, DNA synthesis, and mitosis. These patterns are accompanied by the daily traverse of BMAL-1 protein between the cytoplasm, where it is produced, and nucleus, where it influences timing of cancer cell proliferation. This core clock gene product gates cancer cell proliferation by coordinating clock-controlled proteins, thymidylate synthase [thymidylate synthase activity (TSA) cell DNA replication], WEE-1 (cell mitosis), and vascular endothelial growth factor (growth). 5-Fluorouracil (5-FU)–induced host bone marrow and gut toxicity and tumor shrinkage following administration at six equispaced times of day allowed determination of circadian relationships among tumor growth, relevant clock, and clock-controlled proteins and dependence of 5-FU target availability (TSA) in normal and cancer tissues and resultant 5-FU toxic-therapeutic index. The time of day (hours after lights on) of low TSA in each tissue and tumor is respectively associated with greatest toxicity to that tissue and greatest tumor shrinkage. 5-FU treatment near daily awakening results in least damage to bone marrow and gut, greatest antitumor effect, and best survival. This time of day is associated with maximum tumor nuclear BMAL-1 and total cell WEE-1 protein. The described chain of events, for the first time, links cancer cell clock proteins, cancer cell DNA synthesis, proliferation, TSA, and 5-FU toxic-therapeutic index, explaining the dependence of cancer outcome on circadian timing of 5-FU. [Mol Cancer Ther 2006;5(8):2023–33]

THE CIRCADIAN CLOCK GENE PER1 SUPPRESSES CANCER CELL PROLIFERATION AND TUMOR GROWTH AT SPECIFIC TIMES OF DAY

Cell cycle progression is tightly regulated. The expressions of cell cycle regulators, the products of which either promote or inhibit cell proliferation, oscillate during each cell cycle. Cellular proliferation and the expression of cell cycle regulators are also controlled by the circadian clock. Disruption of the circadian clock may thereby lead to deregulated cell proliferation. Mammalian Per2 is a core clock gene, the product of which suppresses cancer cell proliferation and tumor growth in vivo and in vitro. Because Per1, another key clock gene, is mutated in human breast cancers, and because its clock functions are similar and complementary to those of Per2, we have studied its role in modulating breast cancer cell proliferation and tumor growth. We find that breast cancer growth rate is gated by the circadian clock with two daily peaks and troughs, and that they are coupled to the daily expression patterns of clock-controlled genes that regulate cell proliferation. Down-regulation of the expression of tumor Per1 increases cancer cell growth in vitro and tumor growth in vivo by enhancing the circadian amplitude of the two daily tumor growth peaks. The data of the study suggest Per1 has tumor-suppressor function that diminishes cancer proliferation and tumor growth, but only at specific times of day. (Author correspondence: williamhrushesky@gmail.com).

Daily coordination of cancer growth and circadian clock gene expression

AbstractBackground.Circadian coordination in mammals is accomplished, in part, by coordinate, rhythmic expression of a series of circadian clock genes in the central clock within the suprachiasmatic nuclei (SCN) of the hypothalamus. These same genes are also rhythmically expressed each day within each peripheral tissue. Methods.We measured tumor size, tumor cell cyclin E protein, tumor cell mitotic index, and circadian clock gene expression in liver and tumor cells at six equispaced times of day in individual mice of a 12-h light, 12-h dark schedule. Results.We demonstrate that C3HFeJ/HeB mice with transplanted syngeneic mammary tumor maintain largely normal circadian sleep/activity patterns, and that the rate of tumor growth is highly rhythmic during each day. Two daily 2.5-fold peaks in cancer cell cyclin E protein, a marker of DNA synthesis, are followed by two daily up-to-3-fold peaks in cancer cell mitosis (one minor, and one major peak). These peaks are, in turn, followed by two prominent daily peaks in tumor growth rate occurring during mid-sleep and the second, during mid-activity. These data indicate that all therapeutic targets relevant to tumor growth and tumor cell proliferation are ordered in tumor cells within each day. The daily expression patterns of the circadian clock genes Bmal1, mPer1, and mPer2, remain normally circadian coordinated in the livers of these tumor bearing mice. Bmal1 gene expression remains circadian rhythmic in cancer cells, although damped in amplitude, with a similar circadian pattern to that in normal hepatocytes. However, tumor cell mPer1 and mPer2 gene expression patterns fail to maintain statistically significant daily rhythms. Conclusion. We conclude that, if core circadian clock gene expression is essential to gate tumor cell proliferation within each day, then there may be substantial redundancy in this timing system. Alternatively, the daily ordering of tumor cell clock gene expression may not be essential to the daily gating of cancer cell DNA synthesis, mitosis and growth. This would indicate that host central SCN-mediated neuro–humoro-behavioral controls and/or daily light-induced changes in melatonin or peripherally-induced rhythms such as those resulting from feeding, may be adequate for the daily coordination of cancer cell expression of proliferation related therapeutic targets.