Dale A. Freeman

The low-density lipoprotein pathway of cultured leydig tumor cells ultilization of low-density lipoprotein-derived cholesterol for steroidogenesis

We have previously reported that low-density lipoprotein (LDL) enhances and prolongs steroidogenesis in human choriogonadotropin (CG)-stimulated Leydig tumor cells (MA-10). The studies described herein elucidate the mechanisms by which LDL increases human CG stimulated steroidogenesis. Our results show that the MA-10 cells express the classic LDL pathway. LDL is bound to specific surface binding sites which are regulated by the level of intracellular cholesterol. The cellular processing of bound LDL is temperature-dependent and is inhibited by blocking lysosomal function. By using an LDL derivative in which the core cholesteryl esters have been replaced with [3H]cholesteryl linoleate, we show that LDL cholesterol is rapidly utilized for steroid hormone synthesis. The utilization of LDL cholesterol quantitatively accounts for the LDL-induced augmentation of steroidogenesis. We also show that the addition of LDL to human CG-stimulated MA-10 cells maintains cellular free and esterified cholesterol levels and increases progesterone biosynthesis. The addition of LDL does not, however, affect the cellular utilization of preexisting cholesterol stores for steroidogenesis.

Serum Levels of Squamous Cell Carcinoma Antigen and Ovarian Carcinoma Antigen (CA 125) in Patients with Benign and Malignant Diseases of the Uterine Cervix

In the present study we evaluated the clinical usefulness of the tumor antigens, squamous cell carcinoma antigen (SCC) and ovarian carcinoma antigen (CA 125), in populations of patients with benign and malignant cervical disease. SCC and CA 125 levels were determined in the serum of 59 patients with invasive carcinoma of the uterine cervix and in 21 patients with benign cervical diseases. Before treatment of cervical cancer, SCC levels were elevated in 63% of the patients with squamous cell cancer while all 5 patients with adenocarcinoma had normal levels. CA 125 levels were elevated in 21% of the patients with cervical squamous cell cancer and in 3 of the 5 cases of adenocarcinoma of the cervix. In patients with benign cervical diseases, only 1 had a positive SCC level and none were positive for CA 125. No correlation was found between SCC levels and histological differentiation or clinical stage. In positive patients, serial SCC determinations correlated with the clinical course in 72.2%. Increasing levels were always associated with progression and increased on average 3 months before there was clinical evidence for disease progression. It is concluded from these studies that SCC levels are a useful marker for cervical cancer progression and recurrence. Levels of CA 125 were more likely to be elevated in patients with adenocarcinoma than squamous cell carcinoma, but when elevated in these latter patients, it also tended to predict tumor recurrence.

Constitutive steroidogenesis does not require the actions of cAMP on cholesteryl ester hydrolysis or internalization of plasma membrane cholesterol

The R2C Leydig tumor cells demonstrate constitutive steroidogenesis as well as the constitutive production of the steroidogenic acute regulatory peptide (StAR). Since the introduction of StAR into cells does not by itself reproduce the steroidogenic response of cAMP, the present studies were designed to determine whether processes normally stimulated by cAMP in responsive cells were maximally, constitutively expressed in the R2C cells. Measurements of cholesteryl ester hydrolytic rate and plasma membrane cholesterol internalization revealed that both processes are stimulated by dibutyryl-cAMP in R2C cells. The effect of cAMP on these processes was of the same magnitude as that detected in hormonally responsive cells. Thus, in R2C cells the unstimulated basal rate of either process was sufficient to maintain maximal stimulation of steroidogenesis.

Plasma membrane steroidogenic cholesterol: The relative importance of membrane internalization rate and cholesterol extraction rate of internalized membrane

Most cholesterol destined to become steroid hormone passes through the plasma membrane. Using MA-10 Leydig tumor cells as a model system, we determined that plasma membrane cholesterol disappeared 2.5-fold faster in cAMP-stimulated cells. Stimulation of MA-10 cells increased internalization so that 1.4-fold more membrane cycled through the cell per unit time. Increased internalization accounted for 26% of the cholesterol actually lost from cycling membrane. The remaining 74% was accounted for by increasing the extraction of cholesterol from internalized membrane.

Barbiturates inhibit progesterone synthesis in cultured Leydig tumor cells and human granulosa cells

Screening drugs used in obstetrical practice for effects on steroid hormone synthesis revealed that phenobarbital inhibited progesterone synthesis in MA-10 Leydig tumor cells. The inhibition was apparently a drug class effect since it could be reproduced by other barbiturates. Barbiturate blockade was reversible and could be bypassed in the MA-10 cells by using 22-hydroxycholesterol. Human granulosa cell progesterone synthesis was also inhibited in a dose dependent fashion by phenobarbital, secobarbital and barbituric acid. Significant inhibition occurred in dose ranges that would be therapeutic for treating epilepsy. From these data we conclude that barbiturates block steroidogenesis by inhibiting cholesterol transport to the cholesterol side chain cleavage enzyme.