Combined-modality oncotherapy with cyclophosphamide (NSC-26271) and radiotherapy: control of murine mastocytoma.

We investigated the effects of cyclophosphamide, alone and in combination with a 1,000-R/week radiotherapy schedule, on the growth of solid P815X2 tumors in 12-week-old male DBA/2 mice. Single-dose treatments of 150 mg cyclophosphamide/kg were given to animals bearing tumors of different ages. Such treatment of young tumors resulted in proportionately greater degrees of regression and steeper regrowth curves than did treatment of older tumors. Although slopes of regrowth curves differed greatly, time to regrowth (to pretreatment size) was the same for all age classes of tumors. Graded weekly exposures of 50-250 mg/kg for 4 weeks resulted in dose-dependent increases in incidence of complete remission, duration of remission (time to regrowth), and mean animal life-spans. The combination of radiotherapy to the tumor and 75, 150, or 225 mg cyclophosphamide/kg/week resulted in better local tumor control than occurred with radiotherapy or the drug alone. However, a dose-dependent increase in radiosensitivity of the gastrointestinal mucosa included in radiotherapy fields was observed. A 3-week course of radiotherapy plus 75 mg cyclophosphamide/kg/week (which is tolerated by the mucosa) increased animal lifespans to 165% of those of controls.

Control of local tumor growth with combined fractionated radiotherapeutic and chemotherapeutic regimens.

A treatment concept for the control of tumor growth utilized weekday radiotherapy and weekend chemotherapy. Mice were given sc injections of P815X2 mastocytoma cells on the lower back (day 0) and separated into the following treatment groups: 5-day/week X-irradiation, adriamycin alone at either 5 mg/kg body wt (days 6 and 13) or 2 mg/kg (days 5, 12, and 19), and combined radiotherapy and chemotherapy. Untreated controls had a mean tumor volume of 2.77 cm-3 and a mean survival time of 24 days. Adriamycin alone at 5 mg/kg resulted in an eventual tumor of 70 percent of the control value at death, whereas at 2mg/kg the tumor volume was 60 percent of control. After radiotherapy only, tumor size was 52 percent of control. Irradiation plus either 5 or 2 mg drug per kg body wt resulted in tumor volumes of 23 and 30 percent, respectively, of control values. Although no treatment regimen prolonged survival, the marked reduction in local tumor growth with combination therapy indicates that it may be a useful concept in future cancer therapy.

Influence of adriamycin and adriamycin-radiation combination on jejunal proliferation in the mouse.

The influence of adriamycin and adriamycin-radiation combinations on posttreatment proliferative activity of the mouse jejunum was examined by measuring [3H]thymidine incorporation. Single doses of 5 or 10 mg/kg produced a transient reduction in the proliferative activity, while 1 mg/kg had little effect. After 10 mg/kg, there was a rapid decrease in the number of mitotic figures, followed by a gradual decrease in the number of and rate of DNA synthesis in S-phase cells. A compensatory epithelial hyperplasia characterized by an enlarged crypt proliferative population and shortened mitotic cycle duration was observed beginning 48 hr after treatment. Multiple doses of adriamycin totalling 10 mg/kg inhibited cell production to a greater extent than the equivalent single dose. In combination with 1000 R, adriamycin (5 mg/kg) given from 96 hr before to 72 hr after irradiation reduced the amount of postirradiation proliferation.

Model for the genetic evolution of human solid tumors.

A conceptual model is proposed for the genetic evolution of many human solid tumors that is based on the observations that cancer cells may spontaneously double their chromosome number; that cells with excessive chromosome numbers may be cytogenetically unstable, both losing chromosomes randomly during subsequent cell divisions, and often developing structural abnormalities in the chromosomes that are retained; and that some structural chromosome abnormalities may activate growth-promoting genes. The sequence of tetraploidization with chromosome loss can occur repeatedly in a given tumor. The available evidence supporting the model is reviewed. A computer simulation system that embodies these concepts is described and the model is used to generate distributions of chromosome number/cell under various simulated conditions and in a variety of simulated biological settings. A simulation of the time course of changes in chromosome number per cell that accompany the spontaneous neoplastic transformation of mouse fibroblasts in vitro is described. The best fit to the data was obtained when provision was made for the activation of at least two growth-promoting genes. The conditions for generating discrete aneuploid peaks in cytogenetic and flow cytometric studies were explored; our modeling studies suggest that the activation of a growth promoting gene is required in order to produce a discrete aneuploid peak. Our modeling studies suggest that the overrepresentation of individual oncogene-bearing chromosomes in aneuploid cell lines may require the activation of gene dose-dependent growth-promoting genes and is not likely to occur in cell lines in which at least two copies of each normal chromosome are required for cell survival. Overall, the results obtained using the model are consistent with a wide variety of flow cytometric and cytogenetic studies in human solid tumors.

Karyotypic evolution of a human undifferentiated large cell carcinoma of the lung in tissue culture.

Serial cytogenetic studies were performed on a cell line derived from a pleural effusion from a patient with undifferentiated large cell carcinoma of the lung. The initial sample had a broad range of chromosome numbers per cell, with a hypodiploid/pseudodiploid stem line and a hypotetraploid sideline. A sequence consisting of a doubling of chromosome number per cell followed by chromosome loss was observed repeatedly during 40 culture passages. The presence of metaphase spreads showing evidence of endoreduplication suggested this as a likely mechanism for the doubling of chromosome number per cell. Eleven marker chromosomes were observed in the cells of the primary sample; these markers persisted through all subsequent passages. Chromosomes 1, 2, 6, 7, 8, 11, and 16 were consistently overrepresented; each of these chromosomes was involved in marker formation. Chromosomes 4, 5, 9, 10, 19, 21, and 22 were consistently underrepresented. Every chromosome, either in its normal form and/or as part of a marker, was represented on the average by at least one copy per diploid cell. Eighteen new marker chromosomes were observed during the course of cell cultivation; one of these evolved into a clonal marker over the course of six cell passages. Of the new marker chromosomes that were formed during the observation period, the majority were found in hypotetraploid cells.

Measurement of NK activity by the microcytotoxicity assay (MCA): a new application for an old assay.

Natural killer (NK) cells are spontaneously cytotoxic immune effector cells with the ability to selectively destroy tumor cells without harming normal cells. To perform this function, NK cells utilize two main cytotoxicity pathways, the well known perforin/granzyme-mediated secretory/necrotic killing and the recently defined TNF family ligand-mediated non-secretory/apoptotic killing. The former mechanism is manifested mainly against a few cultured leukemia cell targets, while the latter mediates killing against a large variety of tumor cell targets. Therefore, the biological role and significance of these mechanisms might be different. The NK cell-mediated necrotic killing has been reliably and selectively measured in humans by the standard 4-h 51Cr release assay (CRA) against K562 myeloid leukemia cell targets. However, no standardized high throughput assay is available for testing the NK cell-mediated apoptotic killing. Here, we introduce the modified MCA as a convenient method for measuring perforin/granzyme-independent NK cell-mediated apoptotic killing. The assay is performed in microwells of Terasaki tissue culture microtest plates, using adherent tumor cell targets, which are selectively susceptible to non-secretory/apoptotic killing and resistant to secretory/necrotic killing mediated by NK cells. Target cells are plated in microwells and incubated overnight to adhere to the plastic surface and to regenerate cell surface-bound TNF family receptors. Following this adherence, target cells are co-incubated with freshly isolated human peripheral blood mononuclear leukocytes (PBMNL) or purified subpopulations of immune cells for 24 h in various effector/target (E/T) ratios. During this incubation, dead target cells become non-adherent and are removed by washing the wells. Remaining adherent (viable) target cells are fixed, stained and optically counted. A notable dose-dependent (peak at 200:1 E/T ratio), time-dependent (peak at 24 h of incubation) and donor-dependent killing of tumor cells was consistently and reproducibly induced by PBMNL of normal donors. Using purified subpopulations of immune cells, it was demonstrated that among PBMNL, CD3(-)CD56(+)CD16(+) mature NK cells are the only mediators of tumor cell killing in MCA, as well as in CRA. Comparative studies of NK activity detected by MCA and CRA, performed with PBMNL from normal individuals and breast cancer patients, showed no significant correlation between the cytotoxicities measured in the two assays. In addition, while NK activity measured in CRA was normal in most breast cancer patients, NK activity assessed in MCA was decreased in a large majority of the patients. Thus, MCA is a sensitive NK assay, which is biologically different from CRA, and may be clinically relevant. MCA has also a higher throughput, and is more practical and economical than CRA.

Radiation survival of two human cervical carcinoma cell lines after multifraction irradiation.

PURPOSE: Multifraction irradiation may contribute to radiation therapy treatment failure if selection of radiation resistant subpopulations occurs. We sought to determine whether surviving cells following daily fraction irradiation of two human cervical squamous cell carcinoma lines would express different radiation survival characteristics compared to the unirradiated parent. METHODS AND MATERIALS: A late-passage line (HTB35) and an early-passage line (RECA) received daily 2 Gy x-irradiation. Two new stable HTB35 cell lines were established after 40 and 60 Gy (HTB35-40 and HTB35-60). A single line was established from RECA after 30 Gy (RECA-30). High dose rate (74 cGy/min) acute radiation survival curves were prepared from the three new lines and the unirradiated parents. Potentially lethal damage repair (PLDR) and sublethal damage repair (SLDR) responses were detailed for HTB35, HTB35-40 and HTB35-60. Low dose rate (1.27 cGy/min) survival was measured for HTB35 and HTB35-60. Clones were derived from HTB35 and from HTB35-60 and the surviving fraction at 2 Gy (SF2) values were determined. RESULTS: The two parent lines (HTB35 and RECA) differed in acute radiation survival. The surviving lines following multifraction irradiation (HTB35-40, HTB35-60, and RECA-30) showed no change in acute radiation response compared to the appropriate parent. HTB35-40 and HTB35-60 were repair proficient, demonstrating similar PLDR and SLDR recovery ratios as the parent. Likewise, acute, low dose rate survival of HTB35 and HTB35-60 was similar. Nine clones derived from HTB35 lacked a consistent difference in SF2 compared to the original culture. A single clone of seven derived from HTB35-60 was consistently radiation resistant (SF2 = 0.81 +/- 0.06) compared to the original culture (SF2 = 0.50 +/- 0.09). CONCLUSION: No evidence was obtained that cell lines generated following multiple daily fractions of x-irradiation in vitro possessed acute radiation survival or repair characteristics that were different from the unirradiated parent.

Thyroid hormones in human follicular fluid and thyroid hormone receptors in human granulosa cells.

OBJECTIVE: To demonstrate the presence of thyroid hormone in human follicular fluid (FF) and the binding of antithyroid hormone antibodies in human granulosa cells (GCs). DESIGN: Follicular fluids and GCs collected from women undergoing oocyte retrieval after superovulation. SETTING: In Vitro Fertilization-America/Allegheny General Hospital and Reproductive Sciences Research Laboratories, the Department of Obstetrics and Gynecology, The Medical College of Pennsylvania/Allegheny Campus. MAIN OUTCOME MEASURES: Follicular fluid levels of triiodothyronine (T3) determined by a microparticle enzyme immunoassay and FF levels of thyroxine (T4) determined by a fluorescence polarization immunoassay. Three anti-thyroid receptor antibodies were used to determine the presence of thyroid receptor. The binding of these antibodies in GCs was assessed by fluorescent microscopy and flow cytometry. RESULTS: Both T3 and T4 were present in the FF of eight patients studied. A large majority of the samples of individual fluids fell within the normal range for serum. There was a positive correlation between serum T4 values and FF T4 values. The three antithyroid receptor antibodies showed positive nuclear staining of GCs by fluorescent microscopy. The antibody to all thyroid hormone receptors yielded 35% positive cells by flow cytometry, and the site specific antibody for either the alpha-1 or beta-1 receptors yielded 78% and 44% positive cells, respectively. CONCLUSION: These data demonstrated, for the first time, the presence of T3 and T4 in human FF and the presence of T3 binding sites in human GCs and suggest a role for thyroid hormone in the regulation of human GCs.

Thyroid hormone receptor messenger ribonucleic acid in human granulosa and ovarian stromal cells.

OBJECTIVE: To examine whether mRNA for thyroid hormone receptors alpha and beta is present in human granulosa cells in nonstimulated ovaries. DESIGN: Paraffin-embedded sections of ovaries from normally cycling women were analyzed by in situ hybridization with oligonucleotide probes for thyroid hormone receptors alpha and beta. The sense strand oligonucleotide was used as a control for each of the probes. RESULTS: Granulosa cells from the preovulatory antral follicles examined showed positive staining for both the thyroid hormone receptor alpha and beta probes. Positive staining of ovarian stromal cells also was observed for both probes. CONCLUSION: Thyroid hormone receptor mRNAs are expressed in both granulosa cells and ovarian stromal cells found in nonstimulated ovaries. It is, therefore, conceivable that thyroid hormone may play a direct role in human ovarian physiology.

Damage and recovery assessment of the mouse jejunum to abdominal X-ray and adriamycin treatment.

The influence of adriamycin on the post-irradiation proliferative response of the mouse jejunum was examined. Doses of either 5 or 10 mg/kg of adriamycin administered immediately after abdominal irradiation reduced the LD50/7 days by 300-400 R. Neither dosage of the drug reduced the number of surviving crypts, as measured by the crypt isolation and microcolony techniques, for a given radiation exposure. However, both drug dosages reduced the amount of post-irradiation compensatory hyperplasia, as measured by 3H-thymidine incorporation.

Hypothermia following whole-body heating of mice: effect of heating time and temperature.

We have observed an acute and prolonged lowering of body temperature (hypothermia) following whole body heating (WBH) of mice. This phenomenon of heat-induced hypothermia and the subsequent recovery of normal temperatures have been systematically investigated. The hypothermic period can be characterized by two parameters: Tnadir and a recovery time constant (tau). For treatment temperatures below 41 degrees C and treatment durations of 1 h or less, a mild hypothermia (Tnadir greater than 33 degrees C) and fast recovery (tau less than 1 h) occur. Tnadir and tau vary slightly with treatment temperature and are almost independent of treatment length. At treatment temperatures 41 degrees C and above for up to 1 h, we observed acute hypothermia (Tnadir as low as 28 degrees C) and slow recovery (tau = several hours). This region of prolonged hypothermia is characterized by a rapid change of Tnadir and tau with temperature, and a much less rapid change with treatment duration. The WBH temperature-time range causing prolonged hypothermia is very narrow, and if exceeded results in lethality. Critical lethal temperatures have been estimated for several treatment durations from the time constant data. Post-WBH hypothermia can be minimized by keeping the animals in a 37 degrees C environment. However, we find that neither survivability nor intestinal cell repopulation is enhanced by this procedure.

Influence of thyroxine on human granulosa cell steroidogenesis in vitro.

PURPOSE: The objective of this study was to investigate the influence of thyroid hormone on estradiol and progesterone secretion of human granulosa cells maintained in vitro. METHODS: Granulosa cells were obtained by aspiration of preovulatory follicles of woman undergoing assisted reproductive technology. Ovulation induction was performed with GnRH agonist, hMG, and hCG. RESULTS: Granulosa cells were maintained in vitro in a defined medium with added insulin. Twenty-four-hour estradiol and progesterone secretion into the medium were determined for granulosa cells growing in serum-free medium and in serum-free medium with added T4 in a concentration range of 10(-7) to 10(-11) M. CONCLUSIONS: All concentrations of T4 used produced a statistically significant increase in progesterone secretion (range, 1.39 to 1.60 times the baseline amount). The increase in estradiol secretion reached statistical significance only at a T4 concentration of 10(-8) M (1.24 times the baseline amount).

Augmentation by thyroxine of human granulosa cell gonadotrophin-induced steroidogenesis.

The objective of this study was to investigate the influence of thyroid hormone on gonadotrophin-induced oestradiol and progesterone secretion by human granulosa cells maintained in vitro. Granulosa cells were obtained by aspiration of pre-ovulatory follicles from women undergoing assisted reproductive technology. Ovulation induction was performed with gonadotrophin-releasing hormone agonist, human menopausal gonadotrophin and human chorionic gonadotrophin. Granulosa cells were maintained in vitro in a defined medium with added insulin. Between 48 and 72 h after the initiation of cell culture, oestradiol and progesterone secretion into the medium was determined for granulosa cells growing in serum-free medium with follicle-stimulating hormone (FSH)/luteinizing hormone (LH) and in serum-free medium with FSH/LH and thyroxine added in a concentration range of 10(-10)-10(-7) M. All concentrations of thyroxine used produced a statistically significant increase in oestradiol (range 1.18-1.37 times the amount with FSH/LH alone) and progesterone (range 1.29-1.51 times the amount with FSH/LH alone) secretion.

Mode of growth of the jejunal crypt cells of the rat: an autoradiographic study using double labelling with 3H- and 14C-thymidine in lower and upper parts of crypts.

The frequency distribution of cells through the mitotic cycle in lower and upper portions of jejunal crypts of the rat was examined by the 3H-14C-thymidine double labelling technique. Isolated crypts were cut perpendicular to the longitudinal axis so that the percentage of cells in the lower portion varied from 16 to 74%. The lower and upper portion of the same crypt were squashed separately on one microscope slide and the number of 3H- and 14C-only labelled cells were scored to determine the flow rate into and out of S for the two portions. The mitotic cycle and its phases of the crypt epithelial cells were also determined. For lower portions of crypts which contained less than 40% of the total cell number in that crypt the flow rate into S was about 1-7 times that of the flow rate out of S indicating that nearly every mitosis in this region produced two proliferative daughter cells. As the proportion of cells in the lower part of the crypt increased the quotient of the flow rate into S divided by the flow rate out of S decreased, and approached the steady state value of 1-0 in lower portions containing 60-74% of the cells. For upper portions of crypts which contained less than 40% of the total crypt cells the flow rate into S was about 0-2 times that of the flow rate out of S, indicating that in this region mitoses predominantly produced non-proliferative daughter cells. The results obtained were in good agreement with the model of crypt cell proliferation proposed by Cairnie, Lamerton & Steel (1965b).

ICRF-159 enhancement of radiation response in combined modality therapies. I. Time/dose relationships for tumour response.

The combined effect of the chemotherapeutic agent ICRF-159 and irradiation were evaluated using the Lewis lung tumour (LL). At a daily dose of 25 mg/kg, ICOF given alone prevented the progressive growth of LL. Daily pretreatment also potentiated the effects of radiation (600 rad) on tumour growth, provided the pretreatment kinetics of the tumour permitted a response to radiation alone. Single acute doses of the drug failed to alter the growth of LL, and when combined with radiation failed to enhance the radiation effect. Fractionation of the drug (25 mg/kg; 4 doses at 3h intervals) before irradiation, however, results in immediate effects on tumour growth which are more than additive. The results suggest that a low dose of ICRF-159 for extended periods is more effective in enhancing radiotherapy than a high dose provided acutely.