Targeting an anti-cancer drug to tumors should increase the Area Under the drug concentration-time Curve (AUC) in tumors while decreasing the AUC in normal cells and should therefore increase the therapeutic index of that drug. Anti-tumor drugs typically have half-lives far shorter than the cell cycle transit times of most tumor cells. Tumor targeting, with concomitant long tumor exposure times, will increase the proportion of cells that move into cycle when the drug concentration is high, which should result in more tumor cell killing. In an effort to test that hypothesis, we conjugated a natural fatty acid, docosahexaenoic acid (DHA), through an ester bond to the paclitaxel 2'-oxygen. The resulting paclitaxel fatty acid conjugate (DHA-paclitaxel) does not assemble microtubules and is non-toxic. In the M109 mouse tumor model, DHA-paclitaxel is less toxic than paclitaxel and cures 10/10 tumored animals, whereas paclitaxel cures 0/10. One explanation for the conjugate's greater therapeutic index is that the fatty acid alters the pharmacokinetics of the drug to increase its AUC in tumors and decrease its AUC in normal cells. To test that possibility, we compared the pharmacokinetics of DHA-paclitaxel with paclitaxel in CD2F1 mice bearing approximately 125 mg sc M109 tumors. The mice were injected at zero time with a bolus of either DHA-paclitaxel or paclitaxel formulated in 10% cremophor/10% ethanol/80% saline. Animals were sacrificed as a function of time out to 14 days. Tumors and plasma were frozen and stored. The concentrations of paclitaxel and DHA-paclitaxel were analyzed by LC/MS/MS. The results show that DHA targets paclitaxel to tumors: tumor AUCs are 61-fold higher for DHA-paclitaxel than for paclitaxel at equitoxic doses and eight-fold higher at equimolar doses. Likewise, at equi-toxic doses, the tumor AUCs of paclitaxel derived from i.v. DHA-paclitaxel are 6.1-fold higher than for paclitaxel derived from i.v. paclitaxel. The tumor concentration of paclitaxel derived from i.v. paclitaxel drops rapidly, so that by 16 h it has fallen to the same concentration (2.8 microM) as after an equi-toxic concentration of DHA-paclitaxel. In plasma, paclitaxel AUC after an MTD dose of DHA-paclitaxel is approximately 0.5% of DHA-paclitaxel AUC. Thus, the increase in tumor AUC and the limited plasma AUC of paclitaxel following DHA-paclitaxel administration are consistent with the increase in therapeutic index of DHA-paclitaxel relative to paclitaxel in the M109 mouse tumor model. A phase I clinical study has been completed at The Johns Hopkins Hospital to evaluate the safety of DHA-paclitaxel in patients with a variety of solid tumors. Twenty-one patients have been treated to date. The recommended phase II dose is 1100 mg/m(2), which is equivalent to 4.6 times the maximum approved paclitaxel dose on a molar basis. No alopecia or significant peripheral neuropathy, nausea, or vomiting have been observed. Asymptomatic, transient neutropenia has been the primary side effect. Eleven of 22 evaluable phase I patients transitioned from progressive to stable disease, as assessed by follow-up CT. Significant quality of life improvements have been observed. Thus, DHA-paclitaxel is well tolerated in patients and cures tumors in mice by targeting drug to tumors.

The efficiency of detection of H- and K-ras mutations in 27 CD-1 mouse liver tumors by direct sequencing of polymerase chain reaction (PCR)-amplified DNA isolated from formalin-fixed and paraffin-embedded tissues was compared with that after assay by both NIH 3T3 transfection (followed by sequencing of amplified transformant DNA) and direct sequencing of PCR-amplified DNA isolated from frozen tumors. Some tumor samples were chosen for comparison because they contained ras mutations that were detected by either NIH 3T3 transfection or sequencing of PCR-amplified DNA derived from frozen tumors, but were not detected by both techniques. The efficiency of detecting K-ras mutations was similar for sequencing of amplified fragments derived from both paraffin-embedded tissues and from frozen tumors. However, these two techniques differed in their efficacy for detection of H-ras codon 61 mutations. Furthermore, this difference appeared to be mutation-specific: the sequencing of amplified products from paraffin-embedded tumor tissues allowed increased detection of CAA to AAA mutations but decreased detection of CAA to CTA mutations relative to sequencing of amplified fragments derived from frozen tumor DNA. Direct sequencing of PCR products from paraffin-embedded sections was more sensitive than NIH 3T3 transfection for detection of activated K-ras genes containing codon 13 mutations but less sensitive for detection of activated H-ras genes containing codon 61 mutations. In summary, direct sequencing of amplified DNA from either frozen tumors or formalin-fixed, paraffin-embedded tissues can be more sensitive than NIH 3T3 transfection for detection of codon 13-activated K-ras genes. However, it appears to be less sensitive than NIH 3T3 transfection for detection of certain activating H-ras mutations. Depending upon the questions being asked of the data, each of the methods can provide useful information about ras gene mutations in tumor samples. The apparent differences in sensitivities between the methods is not yet understood, but such differences should be considered in the analysis of data obtained when only one method is used.

Some of our previous results had shown that certain relatively weak carcinogens induced DNA single-strand breaks in rat hepatocytes but only with concomitant cytotoxicity. Stronger carcinogens usually induced breaks at relatively nontoxic concentrations. These observations led us to propose that chronic toxicity may be weakly carcinogenic through a cell-mediated mechanism without the necessity for a compound to directly attack DNA. The proposed mechanism is that one of a cell's responses to sublethal toxicity is to release the contents of some, but not all, of its lysosomes. Since lysosomes contain DNA hydrolases, these could enter the nucleus and induce single- and double-strand breaks in the DNA. Such DNA damage could lead to a malignant phenotype by a variety of mechanisms in those cells that survive. Some initial support for this hypothesis comes from our observation that hypotonic shock causes single-strand breaks in the DNA of mouse L1210 cells and that these breaks can be repaired.

We have recently developed an alkaline elution/rat hepatocyte assay to sensitively measure DNA single-strand breaks induced by xenobiotics in non-radiolabeled rat hepatocytes. Here we have evaluated this assay as a predictor of carcinogenic/mutagenic activity by testing 91 compounds (64 carcinogens and 27 non-carcinogens) from more than 25 diverse chemical classes. Hepatocytes were isolated from uninduced rats by collagenase perfusion, exposed to chemicals for 3 h, harvested, and analyzed for DNA single-strand breaks by alkaline elution. DNA determinations were done fluorimetrically. Cytotoxicity was estimated by glutamate-oxaloacetate transaminase release or by trypan blue dye exclusion. The assay correctly predicted the reported carcinogenic/non-carcinogenic potential of 92% of the carcinogens tested and 85% of non-carcinogens tested. The assay detected a number of compounds, including inorganics, certain pesticides, and steroids, which give false-negative results in other short-term tests. Only 2 rat liver carcinogens were incorrectly identified; the other carcinogens incorrectly identified are weakly or questionably carcinogenic (i.e., they cause tumors only in one species, after lifetime exposure, or at high doses). Some chemicals cause DNA damage only at cytotoxic concentrations; of 16 such compounds in this study, 12 are weak carcinogens suggesting a link between DNA damage caused by cytotoxicity and carcinogenesis. Our data indicate that this assay rapidly, reproducibly, sensitively, and accurately detects DNA single-strand breaks in rat hepatocytes and that the production of these breaks correlates well with carcinogenic and mutagenic activity.

Protein degradation occurs more rapidly in senescent WI-38 cultures than in phase II cultures or in SV-40 transformed WI-38 cells (VA-13). The first differences are found in early phase III, when short lived but not long lived proteins are degraded more rapidly. At the end of phase III long lived proteins are also degraded more rapidly as shown by both intermittent perfusion and approach to equilibrium methods. By both methods the rates of protein degradation for the virally transformed derivative are the same as those for phase II WI-38, implying that transformation has not altered these characteristics of protein degradation. WI-38 cells incorporate canavanine, an analog of arginine, into protein. This analog, as well as p-fluorophenylalanine and azetidine carboxylic acid, accelerates the degradation of proteins labeled with [3H]leucine in their presence but does not alter the degradation rates of proteins prelabeled with [14C]leucine in the absence of the analogs. These results imply that the analogs increase the intracellular degradation rates of proteins because they render them more susceptible to the degradative system. Late phase III WI-38 cells may not selectively catabolize proteins containing canavanine as rapidly as do phase II and VA-13 cells. These results imply that the phase III protein degradative system becomes partially defective, thereby losing its ability to rapidly catabolize altered protein which leads to increased levels of abnormal proteins and decreased cell function.

In an effort to determine whether many different abnormal proteins are present in phase III W138 cells, we have determined the susceptibility to proteolysis of the total proteins isolated from phase II and phase III cultures. The results demonstrate that proteins with increased proteolytic susceptibility can be detected only at the last population doubling of W138 cultures, but not sooner. Such increased proteolytic susceptibility indicates that proteins from terminal phase III cells are modified in relation to proteins from phase II and early phase III cultures. The modification could be due to errors of transcription, translation, or to post-translational alterations that may be fundamental or incidental to the in vitro senescence phenomenon. These results suggest that biochemical studies of phase III WI38 cultures should not overlook the last population doubling.

ABSTRACT Cytochalasin B reversibly inhibits cytoplasmic streaming in both Nitella and Avena cells. Colchicine, on the other hand, has no effect on streaming in either plant; nor does colchicine prevent the recovery of streaming after cytochalasin is withdrawn. The inhibition of protein synthesis by cycloheximide has no effect on either streaming itself or on the recovery of streaming after cytochalasin withdrawal. All this suggests that microfilaments may provide one component of the structure that generates the streaming force and that microtubules play little, if any, role in the process. Ultrastructural studies of Nitella demonstrate that microfilaments are localized at the boundary of the streaming endoplasm and the stationary ectoplasm. Microfilaments are organized in discrete bundles, with possible cross-bridges between individual filaments in each bundle. These bundles are closely associated with the extensive endoplasmic reticulum. Cytochalasin B does not cause ultrastructural changes in Nitella microfilaments as it does in some animal-cell filaments. Since the molecular mechanism of cytochalasin’s action is unknown, there may be no necessary correlation between functional inhibition by the drug and altered microfilament morphology. A model is advanced which proposes that streaming is generated by an interaction between microfilaments and the endoplasmic reticulum.

The developmental patterns for mouse liver and kidney arginase were measured by a sensitive radioactive assay from day 8 of gestation until adulthood. On day 8 high arginase activity is generally distributed throughout early embryos. Then, as development proceeds, the arginase activity drops rapidly in liver and kidney, apparently because of mass increase unaccompanied by net arginase synthesis. Suddenly, on day 12 of gestation in liver and on day 16 in kidney, arginase activity begins to accelerate toward adult values.