Detection of residual murine LPC-1 myeloma cells from bone marrow cell mixture after purging by 4-hydroperoxycyclophosphamide.

Mixtures of BALB/c bone marrow (5 X 10(6) and LPC-1 myeloma (2 X 10(6) cells have been purged in vitro with 100 microM 4-hydroperoxycyclophosphamide (4HC) for 30 min at 37 degrees C. Elimination of tumor cells was assessed by monitoring newly synthesized tumor-specific IgG 2a kappa in vitro and by reinjecting the cells subcutaneously into the hindlegs of mice. Drug-treated LPC-1 cells had no detectable tumor production and did not reproduce tumors. Untreated cells regrew as solid tumors and killed the host within 3-4 weeks. Bone marrow cell suspensions purged of tumor cells were then used to reconstitute lethally pretreated mice injected 24 h earlier with 300 mg/kg cyclophosphamide (CY). Mice injected with CY alone died within 10 days. Those reconstituted with bone marrow cells or with purged bone marrow-tumor cell mixture lived longer than 7 months. Mice reconstituted with untreated bone marrow-tumor cell suspension grew tumors, had detectable tumor-specific IgG 2a kappa in their serum, and died by day 44. These studies demonstrate that the success of myeloma cell purging can be determined by monitoring newly secreted tumor protein and that 4HC successfully eliminates malignant plasma cells in vitro without impairment of normal bone marrow stem cell functions.

Hydroxyethyl starch serum levels in leukapheresis donors measured by modified periodic acid-Schiff staining technique.

Hydroxyethyl starch (HES), a glucopyranose polymer used to enhance the yield of granulocytes during leukapheresis, was detected by periodic acid-Schiff (PAS) staining of 1-microliter samples following electrophoresis on thin agarose film. HES migrated with restricted electrophoretic mobility in the far gamma region, and the intensity of staining was found to be concentration dependent. Densitometer scanning showed a linear curve from 30 mg to 600 mg per dl, although bands were identified at concentrations lower than 30 mg per dl. Sera from 46 leukapheresis subjects showed the same restricted bands. HES measured retrospectively in sera obtained on days 1 to 400 following apheresis ranged from 675 to 30 mg per dl. The clearance of HES from the sera of four donors measured serially appeared biphasic with the first T1/2 of 0.4 to 6.4 days and the second T1/2 of 58 to 240 days. HES also was detectable in urine concentrated 100fold for as long as 7 days following leukapheresis . This simplified, and sensitive PAS glycoprotein staining method can be used to detect and follow HES concentrations in serum and may provide a new tool for the study of HES in leukapheresis donors.

Direct bone resorbing activity of murine myeloma cells.

The cellular mechanism(s) responsible for tumor-associated bone resorption in multiple myeloma remain uncertain. Both in vivo and in vitro evidence is presented for the direct resorption of bone by mouse plasmacytomas. Morphological examination of autopsy specimens from tumor-bearing mice revealed in vivo erosion of bony surfaces at sites of tumor cell-bone matrix apposition. No osteoclastic bone resorptive activity was evident. Using a 45Ca-labelled, devitalized bone explant assay system, mouse myeloma cells caused the release of isotope at levels from 200-300% above control values. Control cells such as normal spleen lymphocytes and liver cells did not resorb bone. Demonstration of the ability of myeloma cells to independently destroy bone is important to the understanding of the causes of and development of chemotherapeutic approaches to myelomatous bone resorption.

Modified technique for periodic acid-Schiff staining of glycoproteins on agarose-film electrophoretograms.

We have modified the periodic acid-Schiff staining technique for glycoproteins for use with thin agarose-film electrophoresis membranes. With this procedure, carbohydrate-rich proteins can be detected with negligible background staining and insignificant staining of nonglycoproteins such as albumin and nonglycosylated Bence Jones proteins (free light chains). On the other hand, carbohydrate-rich M components of immunoglobulins M and A in serum and in cerebrospinal fluid from patients with plasma cell dyscrasia are readily detected. The technique is two- to threefold more sensitive than Ponceau S. Glycoproteins in serum and body fluids can be determined as a routine analytical procedure.

Development and characterization of a cyclophosphamide-resistant mouse plasmacytoma cell line.

Murine LPC-1 plasmacytoma is sensitive to treatment with cyclophosphamide (CY), and animals bearing this tumor in advanced stages can be cured with doses as small as 60 mg/kg. With repeated transfer of LPC-1 cells followed by subcurative CY therapy, we have developed from this CY-sensitive parent line (CY-S) a stable CY-resistant subline (CY-R) that is unaffected by doses as large as 250 mg/kg. Stability of this induced CY resistance in the absence of CY treatment has been demonstrated for greater than 14 transfer generations. The CY-R subline has been compared to the CY-S parent line and shown to be similar with respect to morphology, growth kinetics, survival time, synthesis of IgG 2a kappa M component, and DNA content and distribution. The CY-S parent line responds in vivo to CY analogs (ifosfamide and trofosfamide), as judged by decreased tumor mass and increased survival; the CY-R subline is resistant to the CY analogs. The response to carmustine (BCNU) was tested, and resistance to CY did not predict for response to this alkylating agent since the effect on both CY-S and CY-R lines was equal. The significance of the development of this CY-resistant subline and the implications for future research into the mechanisms of such resistance are discussed.

Metabolism of guanido-labeled (C-14)arginine in rats, mice, and man.

[6-14C]arginine, injected intraperitoneally into normal rats, was cleared from the plasma with biphasic decay kinetics. Urinary excretion was efficient (32% of the 25-muCi dose within the first 24 hr) with no preferential tissue retention. In mice, the effective duration of the radiotracer's availability for protein biosynthesis was less than 30 min. When the tracer was administered i.v. to patients with multiple myeloma, it was similarly cleared from the plasma with biphasic kinetics, and was excreted rapidly in the urine (22% of the dose within the first 24 hr). In patients, the guanido-tagged arginine labeled only tumor M component, and the labeling was most intense in patients who had far advanced disease. Estimated radiation dose to humans from a 100-muCi injection was 10 mrads. These studies demonstrate the fesibility of in vivo labeling with [16 -14C]arginine, with minimal radiation hazard, thus providing a simple, sensitive, and specific method for monitoring the synthesis of the plasmacytoma M component in patients with multiple myeloma.

Temporary disappearance ("eclipse") of LPC-1 plasmacytoma M component synthesis following tumor cell transfer.

The early phase of LPC-1 plasmacytoma development was studied by in vivo labeling with [6-14C]arginine using its M component (immunoglobulin G 2a,kappa) as marker. At a time when M component was not detected or faint by protein staining of electrophoretograms, significant labeling of M component was detectable by autoradiography. Labeling of the M component was fairly constant for the first 10 hr but was markedly decreased from Days 1 to 7. Nadir (0 to 3% of initial 30-min value) was observed on Day 3. Recovery of M component labeling to the 30-min level was complete by Day 13. This period of marked reduction or "eclipse" in newly synthesized M component was shortened by 2 days when mice were pretreated with pristane or cyclophosphamide prior to tumor cell transfer. The eclipse period was also 2 days shorter in athymic BALB/c-nu/nu mice than in normal BALB/c mice. The eclipse period corresponds to the classical "lag" following tumor cell transfer before tumor growth can be detected by conventional methods. The sensitivity of the [6-14C]arginine pulse permits the in vivo detection of small numbers of tumor cells (as few as 10(6) cells) over the early time periods after cell transfer. Modification of eclipse by manipulating host and/or tumor cells may elucidate the accompanying cellular and biochemical events.

Studies with murine LPC-1 plasmacytoma using [6-14C]arginine.

[16-14C]Arginine ([6-14C]Arg) was used as an in vivo pulse label to study BALB/c murine LPC-1 plasmacytoma synthesis and secretion of its tumour-associated M component (IgG2a, kappa). With this isotope, an eight- to ten-fold enhancement in the labelling of the gamma globulin region and ten-fold reduction in the albumin labelling were observed. Production and secretion of the M component was detected (within 30 min) after cell transfer. Only mice which received tumour cells showed significant labelling in the gamma globulin region 24 hr after isotope injection. The labelling behaviour of the tumour M component correlated with the administered cell dose. The peak heights of radioactivity in the gamma region increased with increments in cell number. When the percentage radioactivity diverted into M component was plotted as a function of cell dose, a linear relationship was noted. This study demonstrates the feasibility of using [6-14C]Arg as a tool to follow the newly synthesized tumour-associated protein, and provides a means of estimating tumour cell number.