L. Clifton Stephens

Expression of Autotaxin and Lysophosphatidic Acid Receptors Increases Mammary Tumorigenesis, Invasion, and Metastases

Lysophosphatidic acid (LPA) acts through high-affinity G protein-coupled receptors to mediate a plethora of physiological and pathological activities associated with tumorigenesis. LPA receptors and autotaxin (ATX/LysoPLD), the primary enzyme producing LPA, are aberrantly expressed in multiple cancer lineages. However, the role of ATX and LPA receptors in the initiation and progression of breast cancer has not been evaluated. We demonstrate that expression of ATX or each edg family LPA receptor in mammary epithelium of transgenic mice is sufficient to induce a high frequency of late-onset, estrogen receptor (ER)-positive, invasive, and metastatic mammary cancer. Thus, ATX and LPA receptors can contribute to the initiation and progression of breast cancer.

Permanent radiation myelopathy

Because of the morbidity and mortality associated with radiation myelopathy, radiation injury to the spinal cord has been the subject of more clinical reports than any other normal-tissue injury. The spinal cord is perhaps second only to skin as the organ most frequently studied by radiobiologists. Its response has been investigated in mice, rats, cats, guinea pigs, rabbits, pigs, dogs, and rhesus monkeys—virtually every laboratory animal species. Given this level of attention, it is surprising what is not known about the radiation response of the spinal cord. Relatively few animal studies have been performed on the effect of cytotoxic drugs on the radiation response of the spinal cord, the age effects, the effects of blood pressure or other physiological conditions that are controllable in the laboratory, or the relative response of various anatomical levels of the spinal cord. In addition to these unresolved factors, the dose response in the human is still poorly defined. However, as a result of the att...

Radiation apoptosis of serous acinar cells of salivary and lacrimal glands

Xerostomia and xerophthalmia are common and potentially serious local side effects of radiotherapy for head and neck cancer. Clinical observations supported by experimental findings show that radiation, even in low doses, causes acute diminutions of saliva and tears by rapidly killing the serous cells of the salivary and lacrimal glands, respectively. Serous acini of salivary and lacrimal glands have similar developmental, morphologic, and functional characteristics. Serous acinar cells are functionally mature, secretory epithelial cells that normally do not divide and are long lived. Irradiation of the salivary and lacrimal glands of rhesus monkeys resulted in selective death of serous acinar cells within 24 hours. The paradigm for acute radiation seroadenosis is intermitotic or interphase cell death caused by apoptosis.

EXTENT AND KINETICS OF RECOVERY OF OCCULT SPINAL CORD INJURY

PURPOSE To obtain clinically useful quantitative data on the extent and kinetics of recovery of occult radiation injury in primate spinal cord, after a commonly administered elective radiation dose of 44 Gy, given in about 2 Gy per fraction. METHODS AND MATERIALS A group of 56 rhesus monkeys was assigned to receive two radiation courses to the cervical and upper thoracic spinal cord, given in 2.2 Gy per fraction. The dose of the initial course was 44 Gy in all monkeys. Reirradiation dose was 57.2 Gy, given after 1-year (n = 16) or 2-year (n = 20) intervals, or 66 Gy, given after 2-year (n = 4) or 3-year (n = 14) intervals. Two animals developed intramedullary tumors before reirradiation and, therefore, did not receive a second course. The study endpoint was myeloparesis, manifesting predominantly as lower extremity weakness and decrease in balance, occurring within 2.5 years after reirradiation, complemented by histologic examination of the spinal cord. The data obtained were analyzed along with data from a previous study addressing single-course tolerance, and data from a preliminary study of reirradiation tolerance. RESULTS Only 4 of 45 monkeys completing the required observation period (2-2.5 years after reirradiation, 3-5.5 years total) developed myeloparesis. The data revealed a substantial recovery of occult injury induced by 44 Gy within the first year, and suggested additional recovery between 1 and 3 years. Fitting the data with a model, assuming that all (single course and reirradiation) dose-response curves were parallel, yielded recovery estimates of 33.6 Gy (76%), 37.6 Gy (85%), and 44.6 Gy (101%) of the initial dose, after 1, 2, and 3 years, respectively, at the 5% incidence (D(5)) level. The most conservative estimate, using a model in which it was assumed that there was no recovery between 1 and 3 years following initial irradiation and that the combined reirradiation curve was not necessarily parallel to the single-course curve, still showed an overall recovery equivalent to 26.8 Gy (61%). The spinal cords of symptomatic monkeys consistently revealed a mixture of white matter necrosis and vascular injury, but the majority of spinal cords of asymptomatic animals did not exhibit overt lesions detectable by light microscopy. CONCLUSION Combined analysis with the data of the previous studies yielded firm evidence that the spinal cord has a large capacity to recover from occult radiation injury induced by a commonly prescribed elective dose. This finding strengthens the rationale for selective use of radiotherapy to treat second primary tumors arising in previously irradiated tissues or late recurrences. However, some caution should be exercised in applying quantitative experimental data, because the length of follow-up in these experiments was limited to 2-2.5 years after reirradiation, whereas human myelopathy cases occasionally occur after longer latency. Because there is a large variation in long-term recovery among tissues, the tolerance of other tissues at risk should also be taken into account in prescribing therapy.

Astro research fellowship: Apoptosis as a predictor of tumor response to radiation in Stage IB cervical carcinoma

PURPOSE Levels of apoptosis predict for tumor responsiveness to radiation in various animal systems. To investigate the potential role of apoptosis as a predictor of response in human tumors, a retrospective review was undertaken of patients with adenocarcinoma of the cervix whose primary lesion at presentation measured at least 4 cm and who underwent definitive radiation therapy. A previous report had indicated that roughly half this group of patients should have a long-term relapse free survival. METHODS AND MATERIALS Pretreatment biopsy specimens of 44 patients with Stage IB adenocarcinoma of the cervix, whose primary lesion at presentation measured at least 4 cm in greatest dimension, were scored for apoptosis by two independent investigators without knowledge of the treatment outcome, and the results were averaged. Actuarial methods were used to assess overall survival, disease-free survival, determinate survival, and local control as a function of the baseline level of apoptosis. Patients ranged in age from 21 to 87 years and were treated with definitive radiotherapy between 1964 and 1989. Follow-up for the surviving patients ranged from 1 to 278 months, with a mean of 101 months. RESULTS Patients whose tumors had a baseline level of apoptosis above the median value (2%) had a better overall survival than those with lower levels of apoptosis (p = 0.056). A similar trend for disease-free survival (p = 0.32) and determinate survival (p = 0.27) did not reach statistical significance, perhaps because of the small number of patients. Because only 6 of the 44 patients (13%) had a local tumor failure, it was not possible to establish a correlation between the pretreatment level of apoptosis and the local tumor control by radiation. CONCLUSION The baseline level of apoptosis predicted for survival in patients with Stage IB cervical adenocarcinoma. Further investigation of the measurement of apoptosis as a potential predictive assay is warranted in other human tumor systems.

Target cell and mode of radiation injury in rhesus salivary glands

Morphological alterations of parotid and submandibular salivary glands of rhesus monkeys were studied 1-72 h and 16-40 weeks postirradiation (PI) with single photon doses of 2.5-15.0 Gy, or 10.2 Gy given in 6 fractions. Acute degeneration and necrosis of serous cells in both parotid and submandibular glands were clearly expressed by 24 h PI and occurred in a dose-related fashion. In submandibular glands, doses of 12.5 or 15.0 Gy damaged mucous cells, but to a considerably lesser extent than the serous cells in the same glands. No significant sparing was evident with dose fractionation. These observations demonstrate the unique sensitivity of serous cells which appear to undergo interphase cell death after irradiation. The results also show that late atrophy was the direct result of acute loss of serous acini and reflects a lack of regeneration of acinar cells receiving acute injury.

The novel triterpenoid C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid inhibits metastatic murine breast tumor growth through inactivation of STAT3 signaling

We and others have reported that C-28 methyl ester of 2-cyano-3, 12-dioxoolen-1, 9-dien-28-oic acid (CDDO-Me) effectively inhibits the growth of multiple cancer cell types. Our previous studies indicated that prolonged CDDO-Me treatment inactivated extracellular signal-regulated kinase signaling in acute myelogenous leukemia cells. Whether treatment with CDDO-Me has an earlier effect on other proteins that are important for either signal transduction or oncogenesis is unknown. Constitutively activated signal transducer and activator of transcription 3 (STAT3) is frequently found in human breast cancer samples. Constitutively activated STAT3 was shown to up-regulate c-Myc in several types of cancer and has a feedback effect on Src and Akt. To examine the effects of CDDO-Me on STAT3 signaling in breast cancer, we used the murine 4T1 breast tumor model, which is largely resistant to chemotherapy. In vitro, after treatment of 4T1 cells with 500 nmol/L CDDO-Me for 2 h, we found (a) inactivation of STAT3, (b) inactivation of Src and Akt, (c) 4-fold reduction of c-Myc mRNA levels, (d) accumulation of cells in G(2)-M cell cycle phase, (e) abrogation of invasive growth of 4T1 cells, and (f) lack of apoptosis induction. In in vivo studies, CDDO-Me completely eliminated 4T1 breast cancer growth and lung metastases induced by 4T1 cells in mice when treatment started 1 day after tumor implantation and significantly inhibited tumor growth when started after 5 days. In vivo studies also indicated that splenic mature dendritic cells were restored after CDDO-Me treatment. In summary, these data suggest that CDDO-Me may have therapeutic potential in breast cancer therapy, in part, through inactivation of STAT3.

Liposomal vector mediated delivery of the 3p FUS1 gene demonstrates potent antitumor activity against human lung cancer in vivo

Lung cancer is one of the leading causes of death in the world. The underlying cause for lung cancer has been attributed to various factors that include alteration and mutation in the tumor suppressor genes. Restoration of normal function of the tumor suppressor gene is a potential therapeutic strategy. Recent studies have identified a group of candidate tumor suppressor genes on human chromosome 3p21.3 that are frequently deleted in human lung and breast cancers. Among the various genes identified in the 3p21.3 region, we tested the antitumor activity of the FUS1 gene in two human non-small-cell lung cancer (NSCLC) xenografts in vivo. Intratumoral administration of FUS1 gene complexed to DOTAP:cholesterol (DOTAP:Chol) liposome into subcutaneous H1299 and A549 lung tumor xenograft resulted in significant (P=.02) inhibition of tumor growth. Furthermore, intravenous injections of DOTAP:Chol–FUS1 complex into mice bearing experimental A549 lung metastasis demonstrated significant (P=.001) decrease in the number of metastatic tumor nodules. Finally, lung tumor-bearing animals when treated with DOTAP:Chol–FUS1 complex demonstrate prolonged survival (median survival time: 80 days, P=.01) compared to control animals. This result demonstrates the potent tumor suppressive activity of the FUS1 gene and is a promising therapeutic agent for treatment of primary and disseminated human lung cancer.

Transplantation of normal and ectopic human endometrial tissue into athymic nude mice

Implants or tiny circumscribed nodules of endometrial tissue were found in all female mice given intraperitoneal injections of fragments of human normal (proliferative and secretory) or ectopic (endometrioma) endometrium. Half of these animals received estrogen supplementation and the other half received none. The endometriosis tissue present in these animals at 28 or 56 days after inoculation consisted of glands and stroma with an infiltration of hemosiderin-laden macrophages. Glands in tissue transplants of animals given supplemental estrogen tended to be larger, and the secretory endometrium tended to revert to a proliferative pattern. Palpable nodules at the site of subcutaneous inoculations of proliferative endometrium became undetectable grossly and microscopically within 24 to 32 days, whereas endometrioma tissue remained detectable for up to 70 days and resembled the intraperitoneal tissue microscopically. This study demonstrates that human endometrial tissue can be successfully transplanted into the nude mouse and will retain its basic morphology.

L-asparaginase kills lymphoma cells by apoptosis

Microscopic examination of histological sections of lymph nodes from a canine case of malignant lymphoma at 4 h after treatment with L-asparaginase revealed massive destruction of neoplastic cells by what was consistent with apoptosis morphologically. Apoptosis as the mode of cell death after asparaginase treatment was confirmed in a mouse lymphoma cell line (LY-TH) by the characteristic fragmentation of DNA into oligonucleosome-sized pieces and by the morphological changes consistent with apoptosis following treatment in vitro. Applied to these cells, asparaginase was found to be most cytotoxic over the range of 1–10 IU/ml. Even after 4 h of asparaginase treatment at 100 IU/ml, protein synthesis was reduced by only one-half, yet DNA fragmentation reached 40%. Other agents that affect protein synthesis (cycloheximide and actinomycin D) caused apoptosis as well; however, agents (radiation, prednisolone, and VP-16) whose mechanisms are different from inhibition of protein synthesis also caused apoptosis. As such, it seems unlikely that protein depletion per se and/or the elimination of specific shortlived proteins is the triggering event that leads to cell death. It is more likely that the suspension of cellular proliferation commits cells to apoptosis after asparaginase treatment.