Identification of virus resistant tumor cell subpopulations in three-dimensional uveal melanoma cultures.

To better understand melanoma resistance to herpes simplex virus type 1 (HSV-1)-mediated oncolysis, traditional two-dimensional (2D) cultures and extracellular matrix (ECM) containing three-dimensional (3D) cultures of OCM1 and C918 uveal melanoma cells were infected with an HSV-1 strain that expresses the green fluorescent protein (GFP) marker during replication. Although 2D cultures were completely destroyed within a few days of HSV-1 inoculation, viable GFP-negative tumor cells remained detectable in 3D cultures for several weeks. Tumor cells with increased resistance to HSV-1 included cells that formed vasculogenic mimicry patterns and multicellular spheroids and cells that invaded Matrigel individually. Mechanisms of tumor resistance against HSV-1 in the 3D environment included impaired virus spread in the ECM and ECM-mediated inhibition of viral replication after viral entry into tumor cells. Observations also suggested that HSV-1 established quiescent infection in some tumor cells present in multicellular spheroids and that this could revert to productive viral infection when the tumor growth pattern changed. These findings indicate that 3D tumor cell cultures can be used to identify distinct tumor cell populations with increased resistance to HSV-1 and to explore mechanisms of ECM-mediated tumor resistance to oncolytic virotherapy.

A recycling pathway for resecretion of internalized apolipoprotein E in liver cells.

We have investigated the recycling of apoE in livers of apoE(-)/- mice transplanted with wild type bone marrow (apoE(+/+) --> apoE(-)/-), a model in which circulating apoE is derived exclusively from macrophages. Nascent Golgi lipoproteins were recovered from livers of apoE(+/+) --> apoE(-)/- mice 8 weeks after transplantation. ApoE was identified with nascent d < 1.006 and with d 1.006-1.210 g/ml lipoproteins at a level approximately 6% that of nascent lipoproteins from C57BL/6 mice. Hepatocytes from apoE(+/+) --> apoE(-)/- mice were isolated and cultured in media free of exogenous apoE. ApoE was found in the media primarily on the d < 1.006 g/ml fraction, indicating a resecretion of internalized apoprotein. Secretion of apoE from C57BL/6 hepatocytes was consistent with constitutive production, whereas the majority of apoE secreted from apoE(+/+) --> apoE(-)/- hepatocytes was recovered in the last 24 h of culture. This suggests that release may be triggered by accumulation of an acceptor, such as very low density lipoproteins, in the media. In agreement with the in vivo data, total recovery of apoE from apoE(+/+) --> apoE(-)/- hepatocytes was approximately 6% that of the apoE recovered from C57BL/6 hepatocytes. Since plasma apoE levels in the transplanted mice are approximately 10% of control levels, the findings indicate that up to 60% of the internalized apoE may be reutilized under physiologic conditions. These studies provide definitive evidence for the sparing of apoE and its routing through the secretory pathway and demonstrate that internalized apoE can be resecreted in a quantitatively significant fashion.

Assembly of very low density lipoproteins in mouse liver: evidence of heterogeneity of particle density in the Golgi apparatus.

The assembly of very low density lipoproteins (VLDL) by hepatocytes is believed to occur via a two-step process. The first step is the formation of a dense phospholipid and protein-rich particle that is believed to be converted to VLDL by the addition of bulk triglyceride in a second step. Previous studies in our laboratory led us to hypothesize a third assembly step that occurs in route to or in the Golgi apparatus. To investigate this hypothesis, nascent lipoproteins were recovered from Golgi apparatus-rich fractions isolated from mouse liver. The Golgi fractions were enriched 125-fold in galactosyltransferase and contained lipoprotein particles averaging approximately 35 nm in diameter. These lipoproteins were separated by ultracentrifugation into two fractions: d < 1.006 g/ml and d1.006;-1.210 g/ml. The d < 1.006 g/ml fraction contained apolipoprotein B-100 (apoB-100), apoB-48, and apoE, while the d1.006;-1.210 g/ml fraction contained these three apoproteins as well as apoA-I and apoA-IV. Both fractions contained a 21-kDa protein that was isolated and sequenced and identified as major urinary protein. Approximately 50% of the apoB was recovered with the denser fraction. To determine if these small, dense lipoproteins were secreted without further addition of lipid, mice were injected with Triton WR1339 and [(3)H]leucine, and the secretion of apoB-100 and apoB-48 into serum VLDL (d < 1.006 g/ml) and d1.006;-1.210 g/ml fractions was monitored over a 2-h period. More than 80% of the newly synthesized apoB-48 and nearly 100% of the apoB-100 were secreted with VLDL. These studies provide the first characterization of nascent lipoproteins recovered from the Golgi apparatus of mouse liver. We conclude that these nascent hepatic Golgi lipoproteins represent a heterogeneous population of particles including VLDL as well as a population of small, dense lipoproteins. The finding of the latter particles, coupled with the demonstration that the primary secretory product of mouse liver is VLDL, suggests that lipid may be added to nascent lipoproteins within the Golgi apparatus.

Herpes simplex virus type 1 latency in the murine nervous system is associated with oxidative damage to neurons.

The pathological consequences of herpes simplex virus type 1 (HSV-1) latency in the nervous system are not well understood. To determine whether acute and latent HSV-1 infections of the nervous system are associated with oxidative damage, mice were inoculated with HSV-1 by the corneal route, and the extent of viral infection and oxidative damage in trigeminal ganglia and brain was assessed at 7, 90, and 220 days after inoculation. Abundant HSV-1 protein expression in the nervous system was observed in neurons and non-neuronal cells at 7 days after inoculation, consistent with viral replication and spread through the trigeminal and olfactory systems. Acute HSV-1 infection was associated with focal, neuronal and non-neuronal 4-hydroxy-2-nonenal- and 8-hydroxyguanosine-specific immunoreactivity, indicating oxidative damage. Rare HSV-1 antigen-positive cells were observed at 90 and 220 days after inoculation; however, widespread HSV-1 latency-associated transcript expression was detected, consistent with latent HSV-1 infection in the nervous system. HSV-1 latency was detected predominantly in the trigeminal ganglia, brainstem, olfactory bulbs, and temporal cortex. Latent HSV-1 infection was associated with focal chronic inflammation and consistently detectable evidence of oxidative damage involving primarily neurons. These results indicate that both acute and latent HSV-1 infections in the murine nervous system are associated with oxidative damage.

Subrenal capsule assay for squamous cell carcinoma of the head and neck.

The murine subrenal capsule assay is an in vivo method for determining the responsiveness of solid tumor xenografts to chemotherapeutic agents. It was used in this study for the purposes of constructing growth curves of squamous cell carcinoma of the head and neck and collecting pilot data on the effects that indomethacin and cisplatin have on this malignant condition. Nine of the ten assays performed were evaluable. Indomethacin, cisplatin, or both were used as treatment drugs in each assay. One-millimeter fragments of viable tumor, from patients with head and neck squamous cell carcinoma, were implanted beneath the renal capsules of normal immunocompetent mice. Baseline and posttreatment measurements were made of the xenografts, and response to treatment was determined by comparing changes in tumor sizes of the test and control groups. Results indicated that reduction in tumor sizes occurred in indomethacin-treated mice in four assays and in cisplatin-treated mice in six assays. In addition, two separate growth curves were calculated by plotting the mean change in tumor size in five mice per day on days 3 through 7. In conclusion, the subrenal capsule assay is a relatively rapid and inexpensive assay in which squamous cell carcinoma remains viable and therapeutic agents can be tested. However, clinical trials that use this assay to choose treatment drugs are needed in order to correlate assay results with clinical responses.

Tumor responsiveness of squamous cell carcinoma to cisplatin.

The addition of chemotherapy to planned multiple modality treatment of patients with advanced head and neck squamous cell carcinomas may improve survival rates, but individual tumor response is unpredictable. An assay for determining tumor responsiveness to specific chemotherapeutic agents would facilitate treatment selection. This study used the subrenal capsule (SRC) assay to test responsiveness of squamous cell carcinoma to cisplatin. Tumor xenografts obtained from ten patients were implanted beneath the renal capsules of control and treatment mice. Tumor responsiveness was determined by using the paired Student's t-test to compare base-line and post-treatment measurements in the cisplatin-treated mice. Results indicated six of ten tumors were sensitive to cisplatin. In conclusion, tumor sensitivity to cisplatin and other drugs may be predictable with the SRC assay, but to be clinically useful, studies will be necessary to correlate assay results with responses observed in patients.